The Pesticide Chemist and Modern Toxicology - American Chemical

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The Role of Genetic Toxicology in a Scheme of Systematic Carcinogen Testing GARY M. WILLIAMS and JOHN H. WEISBURGER American Health Foundation, Naylor Dana Institute for Disease Prevention, Valhalla, NY 10595 DAVID BRUSICK Director of Genetics and Cell Biology, Litton Bionetics, Inc., Kensington, MD 20795

Progress has been great in recent years in developing means for the evaluation of the hazardous potential of chemicals. A major achievement has been the introduction of in vitro techniques for the rapid identification of mutagenic or carcinogenic potential. However, no single test has been found to be sufficient for detection of all hazardous chemicals and therefore, agreement now exists that a battery of tests is required for toxicological evaluations in mass-screening programs (1-11). Over 100 short-term tests for detecting potential chemical carcinogens and mutagens are available (12) and the critical issue in developing a battery of tests is to formulate appropriate criteria for selecting the best combination of tests. One of the first proposals for the systematic application of short-term tests for the detection of carcinogens and mutagens was that of Bridges (1), in which a three tiered protocol involving submammalian tests, whole mammal tests, and finally, in vivo tests for risk assessment was recommended. A similar approach was developed by Flamm (2), for mutagenicity testing, noting that no single genetic test could detect all genetic events of possible hazard to humans. Whereas Bridges favored tests for chromosomal damage, Flamm, and in a later modification, Green (4), recommended the dominant lethal test. It is interesting that none of these early proposals included a DNA damage test. A hierarchical approach described by Bora (3) did include a DNA damage test, but not Drosophila mutagenesis, which how 0097-6156/81/0160-0057$07.75/0 © 1981 American Chemical Society Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

THE

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PESTICIDE CHEMIST AND MODERN TOXICOLOGY

e v e r , was r e c o m m e n d e d by b o t h B r i d g e s a n d Flamm. Instead, Bora proposed the use of host-mediated systems. Thus, a l l these p r o p o s a l s i n c l u d e t e s t s i n v o l v i n g e f f e c t s i n w h o l e o r g a n i s m s . The rationale f o r t e s t s o f t h i s type has r e c e n t l y been r e v i e w e d by R i n k u s a n d L e g a t o r ( 1 3 ) . W i t h the awareness t h a t t e s t i n g schemes were becoming i n c r e a s i n g l y complex and e x p e n s i v e , r e cent efforts have been initiated to reduce the number o f t e s t s t o a n e s s e n t i a l c o r e ( 5 , 6 , 8 , 9 - 1 1 , 14). C o n s i d e r a t i o n o f the v a r i o u s t e s t i n g schemes t h a t have been p r o p o s e d r e v e a l s t h a t most tiers or batteries are structured around seven tests (10): b a c t e r i a l mutagenesis, eukaryote mutagenesis, D r o s o p h i l a mutagenesis, mammalian c e l l mutag e n e s i s , DNA damage, c h r o m o s o m e damage and m a l i g nant transformation. The emphasis on specific t e s t s a p p e a r s t o be m a i n l y a f u n c t i o n o f w h e t h e r t h e p r o p o s e d scheme i s d i r e c t e d toward o n l y mutag e n t e s t i n g , o r c a r c i n o g e n and m u t a g e n d e t e c t i o n . For example, a l l o f the t e s t s , e x c e p t t r a n s f o r m a t i o n , are l i s t e d by t h e I n t e r n a t i o n a l Commission for P r o t e c t i o n a g a i n s t E n v i r o n m e n t a l M u t a g e n s and Carcinogens (1^5) a s among the most w i d e l y used t e s t s f o r mutagen s c r e e n i n g . Thus, t h e r e i s s u b s t a n t i a l consensus regarding the most u s e f u l t e s t s f o r s c r e e n i n g f o r mutagenicity and carcinogenicity. As y e t , h o w e v e r , such concepts are reflected in current testing r e q u i r e m e n t s t o o n l y a l i m i t e d d e g r e e (16 ) . Criteria

for a Battery

The p h i l o s o p h y u n d e r l y i n g a b a t t e r y i s t h a t a g r o u p o f t e s t s s h o u l d be p e r f o r m e d b e f o r e a d e c i s i o n i s made r e g a r d i n g t h e p o t e n t i a l h a z a r d o f t h e chemical. Thus, a b a t t e r y corresponds to the i n i t i a l " d e t e c t i o n " phase t h a t i s p a r t o f most tiers (1,2J. The crucial difference with a battery, however, i s t h a t i t attempts t o combine "detect i o n " and t h e n e x t s t e p o f a t i e r , " c o n f i r m a t i o n " i n one s t a g e . Implicit i n t h i s approach i s the concept that a l l available short-term tests may yield false positive or false negative results that require parallel data for interpretation. The battery approach thus formally incorporates the c o n c e p t t h a t no d e c i s i o n on p o t e n t i a l hazard s h o u l d be made w i t h o u t t h e minimum d a t a o f f e r e d by the b a t t e r y . The choice of tests to comprise a battery

Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

6.

WILLIAMS ET AL.

Genetic

Toxicology

in Carcinogen

Testing

will v a r y d e p e n d i n g upon w h e t h e r t h e g o a l i s t o d e f i n e p o t e n t i a l mutagens o r c a r c i n o g e n s . Little i s known a b o u t t h e v a l i d i t y o f m u t a g e n i c i t y batt e r i e s b e c a u s e few c h e m i c a l s h a v e b e e n shown t o be m u t a g e n i c t o germ c e l l s in experimental animals, a n d no c h e m i c a l s a r e known t o p r o d u c e human g e r m i nal mutations. Thus, at present, mutagenicity batteries must be constructed to identify the b r o a d e s t p o s s i b l e s p e c t r u m o f g e n e t i c damage ( 1 7 ) . In contrast, carcinogenicity batteries can be verified against in vivo data, albeit with an important qualification. Several lines of evid e n c e now indicate t h a t c a r c i n o g e n s may operated by a variety of mechanisms (14'18*19)· Among t h e s e , g e n o t o x i c e f f e c t s c a n be r e a d i l y detected in short-term tests. Other oncogenic mechanisms of a presumed e p i g e n e t i c n a t u r e are c l e a r l y not detected i n tests with a genetic end-point. Some t e s t s such as m a l i g n a n t t r a n s f o r m a t i o n and sister chromatid exchange, which can be produced by e f f e c t s o t h e r t h a n a d i r e c t a t t a c k o n DNA, may be capable of d e t e c t i n g non-genotoxic carcinogens. I n a d d i t i o n , e f f o r t s a r e b e i n g made t o d e v e l o p i n vitro tests for identifiying the promoting class of e p i g e n e t i c agents (20-23). As y e t , however, none o f t h e s e approaches can be recommended for routine inclusion in a battery. T h e r e f o r e , i n the use of b a t t e r i e s for identification of c a r c i n o g e n s , i t m u s t be r e c o g n i z e d t h a t a w h o l e c a t e g o r y of c h e m i c a l s , c o n t a i n i n g such a g e n t s as s a c c h a r i n , hormones, certain organochlorine compounds and p e s t i c i d e s , a n d s e v e r a l p h a r m a c e u t i c a l s may n o t be detected. Several other principles should guide the construction of a battery. I m p o r t a n t l y , the t e s t s s h o u l d be r e l i a b l e a n d o f c l e a r b i o l o g i c significance (24) . T h i s means t h a t they should truly measure what t h e y p u r p o r t t o measure and t h a t the end point should have conceptual relevance to mutagenicity or carcinogenicity. Secondly, a batt e r y should seek to maximize the m e t a b o l i c parameters provided by a l l tests. As an example, tests with intact cell metabolism should be included to extend the m e t a b o l i s m o b t a i n e d with t h e commonly u s e d exogenous s u b c e l l u l a r preparations. T h i s may be o f p a r t i c u l a r importance in view o f the a r t i f a c t u a l enhancement o f a c t i v a t i o n o v e r d e t o x i f i c a t i o n t h a t i s known t o be c h a r a c t e r i s t i c o f enzyme p r e p a r a t i o n s (25r2J>)* Moreover, t h e DNA adducts f o r m e d by a c t i v a t i o n t h r o u g h mic-


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r o s o m e s d i f f e r f r o m t h o s e p r o d u c e d by i n t a c t cell metabolism (27). Adhering to these concepts, a battery of short-term t e s t s was proposed by W e i s b u r g e r and Williams (5,14,28) as p a r t o f a " d e c i s i o n p o i n t approach. "The

Decision

Point

Approach"

The D e c i s i o n P o i n t A p p r o a c h c o n s i s t s o f five sequential steps i n the e v a l u a t i o n o f the p o t e n tial carcinogenicity of chemicals. (Table 1). T h i s a p p r o a c h was formulated to incorporate into chemical e v a l u a t i o n s e v e r a l newer d e v e l o p m e n t s i n chemical carcinogenesis. Of prime importance among t h e s e was the c o n c e p t t h a t c h e m i c a l s could p r o d u c e an i n c r e a s e i n t h e t u m o r i n c i d e n c e i n e x posed animals, i . e . be carcinogenic, by several d i s t i n c t mechanisms each h a v i n g different theore t i c a l and p r a c t i c a l i m p l i c a t i o n s . One of these mechanisms, p r o p o s e d by Miller and Miller (29), was through the generation of an electrophilic r e a c t a n t which would r e a c t c o v a l e n t l y with cellul a r macromolecules. The work i n s e v e r a l laborat o r i e s ( s e e 14 and 30 f o r r e f e r e n c e s ) h a s s t r o n g l y i n d i c a t e d t h a t DNA i s the c r i t i c a l target. However, in addition to chemicals of this type, others lacking this property were nevertheless carcinogenic or oncogenic. Among c h e m i c a l s o f t h e latter type were p l a s t i c s , hormones, immunosuppressants, cytotoxic agents, co-carcinogens and promoters. T h u s , i t was suggested that chemical carcinogens c o u l d be d i v i d e d i n t o two m a i n c a t e g o r i e s , b a s e d u p o n t h e i r c a p a c i t y t o damage DNA. C a r c i n o g e n s t h a t r e a c t e d c o v a l e n t l y w i t h DNA were c a t e g o r i z e d as g e n o t o x i c , w h i l e t h o s e l a c k i n g t h i s property and probably a c t i n g by o t h e r mechanisms were c a t e g o r i z e d as e p i g e n e t i c (14). The genot o x i c c a t e g o r y thereby c o n t a i n s the c l a s s i c o r g a n ic carcinogens that damage DNA either through d i r e c t chemical r e a c t i v i t y or f o l l o w i n g metabolism by enzyme s y s t e m s (Table 2). In a d d i t i o n , the availability o f some e v i d e n c e f o r DNA damage by inorganic carcinogens l e d to t h e i r placement in this category. The second category, epigenetic carcinogens, i s composed o f t h o s e a g e n t s t h a t have n o t b e e n f o u n d t o damage DNA, but r a t h e r appear to act through o t h e r i n d i r e c t mechanisms ( T a b l e 2 ) . The d e c i s i o n p o i n t approach takes these two categories or types of carcinogens i n t o account i n


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Table

Genetic

I.

Toxicology

in Carcinogen

Testing

D e c i s i o n P o i n t Approach Carcinogen E v a l u a t i o n

61

to

a

A.

Structure

Β.

Γη v i t r o s h o r t - t e r m tests 1. B a c t e r i a l mutagenesis 2. DNA r e p a i r 3. Mammalian m u t a g e n e s i s 4. S i s t e r c h r o m a t i d exchange 5. Cell transformation

1

B .

Decision

C.

Limited i n vivo bioassays 1. S k i n tumor i n d u c t i o n i n mice 2. Pulmonary tumor i n d u c t i o n i n mice 3. Breast cancer i n d u c t i o n i n female Sprague-Dawley rats 4. A l t e r e d f o c i induction i n rodent 1 iver

1

a

of chemical

point:

C .

Decision

point.

D.

Chronic

bioassay

E.

Final

evaluation:

From W e i s b u r g e r

t e s t s u n d e r A a n d B.

t e s t s u n d e r A , B, a n d C

a l l tests.

and W i l l i a m s ( 1 4 ) . Macmillan Publishing Co., Inc.



62

Table

II.

C l a s s i f i c a t i o n of Carcinogenic Chemicals 0

Category

A.

Genotoxic

Carcinogens

1.

Activation-independent

Alkylating agent

2.

Activation aromatic

Polycyclic

3a Β.

Example

and C l a s s

dependent

Hydrocarbon, Nitrosamine Metal

Inorganic^

Epigenetic

Carcinogens

3b

Inorganic^

Metal

4.

Solid

Plastics

5.

Hormone

State

Estrogen Androgen

6.

7.

Purine

Immunosuppressor analog

Phorbol ester

Co-carcinogen

Catechol 8.

Phorbol ester

Promoter

Bile

acid

a

a f t e r Weisburger

b

some a r e t e n t a t i v e l y c a t e g o r i z e d a s g e n o t o x i c because o f e v i d e n c e f o r damage o f DNA; o t h e r s may o p e r a t e t h r o u g h e p i g e n e t i c mechanisms such a s a l t e r a t i o n s i n f i d e l i t y o f DNA polymerases.

and W i l l i a m s

(14)


6.

WILLIAMS ET AL.

Genetic

Toxicology

in Carcinogen

Testing

two ways; (1) a b a t t e r y of s h o r t term t e s t s i s constructed b a s e d u p o n an e f f o r t to i n c l u d e systems t h a t may respond to e p i g e n e t i c as well as genotoxic carcinogens; and (2) i t is formally recognized that a l l types of subchronic testing may fail to detect chemicals that can induce tumors i n a n i m a l s under s p e c i f i c c o n d i t i o n s upon chronic administration. O t h e r e l e m e n t s o f the d e c i s i o n p o i n t a p p r o a c h a r e the use o f a b a t t e r y o f s h o r t term t e s t s t h a t e i t h e r may e l i m i n a t e the need f o r f u r t h e r t e s t i n g o f t h e c h e m i c a l o r may e n a b l e the v e r i f i c a t i o n o f carcinogenic p o t e n t i a l i n one of four l i m i t e d i n vivo bioassays for carcinogenicity. The battery a l s o adds e s s e n t i a l i n f o r m a t i o n f o r data evaluat i o n when a n a l r e a d y c o m p l e t e d c h r o n i c t e s t s e r i e s has y i e l d e d ambiguous r e s u l t s . The decision point approach therefore is a systematic a p p r o a c h to the r e l i a b l e e v a l u a t i o n of the p o t e n t i a l c a r c i n o g e n i c i t y of c h e m i c a l s which p r o v i d e s a framework i n which t o m i n i m i z e the nece s s a r y t e s t i n g i n c h e m i c a l e v a l u a t i o n , and a t the same t i m e d e v e l o p a n u n d e r s t a n d i n g o f t h e m e c h a n ism o f a c t i o n o f a t e s t chemical. As shown i n T a b l e 1, the d e c i s i o n p o i n t approach involves a systematic step-wise progression of tests. A critical evaluation of information obtained and i t s s i g n i f i c a n c e i n r e l a t i o n to the t e s t i n g o b j e c t i v e i s p e r f o r m e d a t t h e end o f e a c h phase. A d e c i s i o n i s made w h e t h e r t h e d a t a available are s u f f i c i e n t to reach a d e f i n i t i v e conclus i o n or whether a higher l e v e l of tests is required. Attention i s paid to q u a l i t a t i v e - p o s i tive or negative - e f f e c t s , and t o q u a n t i t a t i v e h i g h , medium, o r low - e f f e c t s . A.

Structure

of

Chemical

F o r a number o f r e a s o n s , t h e e v a l u a t i o n begins with a c o n s i d e r a t i o n of s t r u c t u r e . Of principal i m p o r t a n c e i s the f a c t t h a t p r e d i c t i o n s as t o w h e t h e r o r n o t a g i v e n c h e m i c a l m i g h t be carcinogenic can be made w i t h fair success within certain classes of chemicals (2_8)· Structure, h o w e v e r , m u s t a l w a y s be c o n s i d e r e d in relationship to species metabolic parameters. The guinea p i g , f o r e x a m p l e , d i f f e r s f r o m o t h e r r o d e n t s o r humans, i n t h a t i t has o n l y l i m i t e d amounts o f the necessary enzymes to carry out N-hydroxylation, and thus m e t a b o l i z e s a r o m a t i c amines almost e x c l u s i v e -


64


ly to d e t o x i f i e d m e t a b o l i t e s . Consequently, the a r y l a m i n e s so f a r t e s t e d i n t h i s s p e c i e s have not been carcinogenic. Other examples of species selectivity based upon m e t a b o l i c c a p a b i l i t y are w e l l documented (30). I n f o r m a t i o n o n s t r u c t u r e and m e t a b o l i s m a l s o p r o v i d e s a g u i d e t o t h e s e l e c t i o n among l i m i t e d b i o assays at stage C and, as more information a c c r u e s , may e v e n t u a l l y c o n t r i b u t e t o s e l e c t i o n o f s p e c i f i c s h o r t - t e r m t e s t s a t s t a g e B. Β.

In V i t r o

Short-term

Tests

No i n d i v i d u a l s h o r t - t e r m t e s t t h a t has been studied adequately has detected a l l carcinogens t e s t e d , or even a l l g e n o t o x i c c a r c i n o g e n s . Thus, based on this fact alone, a battery of tests i s necessary. However, the i m p o r t a n c e of a b a t t e r y becomes o b v i o u s upon c o n s i d e r a t i o n o f the c o m p l e x i t y o f m e t a b o l i s m a n d m e c h a n i s m o f a c t i o n o f chem i c a l carcinogens. As i n d i c a t e d e a r l i e r , c a r c i n o g e n s c a n be c l a s s i f i e d a s g e n o t o x i c o r e p i g e n e t i c . Most i n v i t r o tests identify genetic effects and thus d e t e c t o n l y g e n o t o x i c c a r c i n o g e n s . If epi g e n e t i c c a r c i n o g e n s a r e t o be d e t e c t e d , a d d i t i o n a l tests w i l l have t o be developed. Known species d i f f e r e n c e s i n response t o c a r c i n o g e n s c a n be r e lated to a large extent (but not e x c l u s i v e l y ) t o m e t a b o l i s m and t h u s , t e s t s w i t h d i f f e r e n t metabol i c c a p a b i l i t i e s are important. A s c r e e n i n g b a t t e r y must include microbial mutagenesis tests, because these have been the most s e n s i t i v e , effective, and readily performed screening tests thus far (31-3_3) . In d e c i d i n g what o t h e r t e s t s to i n c l u d e , i t i s important to c o n s i d e r the c o n t r i b u t i o n o f the p r o p o s e d test in terms of metabolic capability, reliability, and biological significance of the end point. The bacterial mutagenesis tests require a mammalian enzyme p r e p a r a t i o n t o p r o v i d e f o r metabolism of p r o c a r c i n o g e n s a n d h e n c e , any t e s t t h a t i s d e p e n d e n t upon an enzyme p r e p a r a t i o n d o e s n o t e x p a n d t h e metabolic capability of the battery since this f a c t o r i s the l i m i t i n g p a r t o f a t e s t s e r i e s . M u t a g e n e s i s o f m a m m a l i a n c e l l s i s recommended f o r i n c l u s i o n i n the b a t t e r y b e c a u s e i t has a d e f i n i t i v e end p o i n t l i k e b a c t e r i a l m u t a g e n e s i s , b u t involves effects on the more highly organized e u k a r y o t i c genome (3_4,35). Moreover, d i f f e r e n c e s in the mutagenic r e s p o n s e between m i c r o b i a l and mammalian c e l l s h a v e b e e n o b s e r v e d (36).


6.

WILLIAMS ET AL.

Genetic

Toxicology

in

Carcinogen

Testing

Tests f o r DNA damage o r chromosome effects p r o v i d e f u r t h e r e v i d e n c e o f t h e a b i l i t y o f a chemi c a l to a l t e r genetic material. Proposed i n d i c a t o r s f o r DNA damage t e s t s i n c l u d e DNA fragmentation (37), i n h i b i t i o n of DNA synthesis (38), and DNA repair (39). Measurement of DNA fragmentation, although showing an excellent correlation with carcinogenicity has a conceptual disadvantage in t h a t DNA d e g r e d a t i o n can o c c u r as a r e s u l t o f cell death. I n h i b i t i o n o f DNA s y n t h e s i s has been suggested to o f f e r an advantage in its ability to d e t e c t i n t e r c a l a t i n g a g e n t s (38). T h i s may not be a s u b s t a n t i a l advantage because pure intercalators are a limited group of chemicals comprising at b e s t weak c a r c i n o g e n s and DNA synthesis is also inhibited by noncarcinogenic intercalators. Furthermore, the intercalating agents with react i v e g r o u p i n g s do i n d u c e DNA repair. DNA r e p a i r i s a s p e c i f i c r e s p o n s e t o DNA damage and unlike DNA fragmentation and inhibition c a n n o t be a t t r i b u t e d to t o x i c i t y (40). Therefore, DNA r e p a i r t e s t s o f f e r an end p o i n t o f h i g h specificity biologic significance and are recommended in preference to these other assays. A chromosomal t e s t i s i n c l u d e d i n the battery to p r o v i d e d e t e c t i o n of chemical e f f e c t s at the highest l e v e l of genetic organization (13). In addition, such tests may detect nongenotoxic agents that operate through other e f f e c t s involvi n g p r o c e s s e s s u c h a s DNA r e p l i c a t i o n and chromosome s e p a r a t i o n . C e l l t r a n s f o r m a t i o n i s included because t h i s alteration is potentially the most relevant to carcinogenesis. H o w e v e r , much more n e e d s t o be done t o c l a r i f y the significance and limitations o f t h i s end point. 1.

Bacterial

Mutagenesis

V a l u a b l e b a c t e r i a l s c r e e n i n g t e s t s have been developed in the laboratories of Ames (41) and Rosenkranz (42) . The Ames test measures back mutation to histidine independence of histidine mutants of Salmonella typhymurium and can be conducted with strains that are also repair d e f i c i e n t , t h a t p o s s e s s a b n o r m a l i t i e s i n the cell wall to make them p e r m e a b l e to carcinogens, or that carry an R factor enhancing mutagenesis. Hence, these o r g a n i s m s are highly susceptible to


THE

66


m u t a g e n e s i s m a k i n g them s e n s i t i v e i n d i c a t o r s . The t e s t d e v e l o p e d by R o s e n k r a n z a n d a s s o c i a t e s utilizes DNA repair-deficient Escherichia coli and measures their enhanced susceptibility to cell k i l l i n g by c a r c i n o g e n s . I n t h i s system, a chemical t h a t i n t e r a c t s w i t h DNA i s more t o x i c t o t h e r e p a i r - d e f i c i e n t s t r a i n than t o w i l d type E . c o l i because t h e m u t a n t s t r a i n c a n n o t r e p a i r t h e damage. T h u s , by m e a s u r e m e n t o f r e l a t i v e t o x i c i t y a n i n d i c a t i o n o f DNA i n t e r a c t i o n i s o b t a i n e d . These t e s t s a r e d e p e n d e n t u p o n mammalian enzyme p r e p a r a tions f o rmetabolism of carcinogens. The c a p a b i l i t y o f t h e Ames t e s t t o d e t e c t c e r t a i n c a r c i n o g e n s h a s b e e n e n h a n c e d by a p p l i c a t i o n o f p r e i n c u b a t i o n of t h e compound and t h e b i o c h e m i c a l activation s y s t e m w i t h t h e t e s t o r g a n i s m (4_3). 2,

Mammalian

Mutagenicity Tests

The three mutational assays i n mammalian c e l l s t h a t have been most w i d e l y used f o r c a r c i n o gen screening are resistance to purine analogs (44-46), bromodeoxyuridine (BUdR) ( 4 7 ) o r o u a b a i n (48). Of t h e s e , p u r i n e a n a l o g r e s i s t a n c e i s t h e most p o p u l a r . I n t h i s assay, mutants l a c k i n g the p u r i n e s a l v a g e pathway enzyme hypoxanthine-guanine p h o s p h o r i b o s y l t r a n s f e r a s e a r e i d e n t i f i e d by t h e i r resistance to toxic purine analogs such as 8-azaguanine o r 6-thioguanine that k i l l c e l l s that u t i l i z e the analogs. T h i s assay has the advantage over ouabain r e s i s t a n c e t h a t i t i n v o l v e s a noness e n t i a l f u n c t i o n , u n l i k e t h e membrane A T P a s e s y s tem involved i n ouabain resistance, and hence t h e r e a r e no l e t h a l m u t a n t s . I t s advantage over the measurement of thymidine kinase-deficient m u t a n t s by r e s i s t a n c e t o BUdR i s t h a t t h e g e n e f o r hypoxanthine-guanine phosphoribosyl transferase i s on t h e X-chromosome r a t h e r t h a n a s o m a t i c chromosome, a s w i t h t h y m i d i n e k i n a s e . C o n s e q u e n t l y , o n l y one f u n c t i o n a l c o p y i s p r e s e n t i n e a c h c e l l a n d , as a r e s u l t , m u t a t i o n s i n w i l d t y p e c e l l s c a n be measured, whereas a heterozygous mutant is required f o r measurable mutation t o homozygous t h y m i d i n e k i n a s e d e f i c i e n c y a n d BUdR r e s i s t a n c e . The t a r g e t c e l l s used i n p u r i n e a n a l o g r e s i s tance a s s a y s have a l m o s t a l l been f i b r o b l a s t - l i k e , s u c h a s t h e V 7 9 a n d CHO l i n e s , w h i c h h a v e displayed little ability to activate carcinogens, other than p o l y c y c l i c aromatic hydrocarbons ( 4 5 ) . This deficiency has been overcome by providing


6.

WILLIAMS ET AL.

Genetic

Toxicology

in Carcinogen

Testing

exogenous metabolism mediated by e i t h e r cocultivated c e l l s ( 4 5 , 4 9 ) o r enzyme p r e p a r a t i o n s (44) . T h e l a t t e r a g a i n o f f e r s no e x t e n s i o n i n m e t a b o l i c c a p a b i l i t y over t h a t used for bacterial systems. However, t h e use o f f r e s h l y isolated hepatocytes as a f e e d e r system (4_9) o f f e r s a d d i t i o n a l possibilities s i n c e the metabolism of hepatocytes has been shown t o be different than that of liver enzyme p r e p a r a t i o n s ( 2 6 , 2 7 ) . Another potentially u s e f u l development i s the f i n d i n g t h a t l i v e r epithelial cultures can be mutated by activationd e p e n d e n t c a r c i n o g e n s (4_6) a n d may therefore prov i d e another system with i n t a c t c e l l metabolism. 3.

DNA

Repair

The covalent interaction of chemicals with DNA provokes an e n z y m a t i c r e p a i r o f the damaged r e g i o n s o f DNA known a s e x c i s i o n r e p a i r . Two types of e x c i s i o n repair are r e c o g n i z e d , base removal and n u c l e o t i d e r e m o v a l (5_0) . The f i r s t s t e p i n each of these d i f f e r s , but both p r o c e s s e s result i n i n c i s i o n o f t h e s t r a n d o f DNA near the p o i n t of damage and excision by an endonuclease of a s t r e t c h o f DNA i n t h e damaged r e g i o n . The gap i s then f i l l e d by r e s y n t h e s i s o f a p a t c h u s i n g the opposite s t r a n d as a template and the patch is c l o s e d by a l i g a s e . S e v e r a l of these steps could be m e a s u r e d a s a n indication of repair, but the r e s y n t h e s i s o f the p a t c h i s most w i d e l y used to monitor r e p a i r i n s c r e e n i n g systems. Repair synt h e s i s c a n be m e a s u r e d i n a v a r i e t y o f w a y s (40, 50). Several of the definitive procedures are technically sufficiently demanding so that they have not received much attention for screening purposes. Of the s i m p l e r p r o c e d u r e s available, autoradiographic measurement o f r e p a i r s y n t h e s i s has the a d v a n t a g e o v e r l i q u i d s c i n t i l l a t i o n c o u n t ing i n that i t excludes c e l l s i n r e p l i c a t i v e synt h e s i s , whereas these are p a r t of the background with l i q u i d s c i n t i l l a t i o n counting. In a d d i t i o n , with l i q u i d scintillation counting, increases i n i n c o r p o r a t i o n can r e s u l t from changes i n uptake or the pool size of thymidine without any repair occurring. Furthermore, autoradiography permits a d e t e r m i n a t i o n o f the f r a c t i o n o f c e l l s responding i n the a f f e c t e d p o p u l a t i o n . Two a d d i t i o n a l compl i c a t i o n s w i t h most r e p a i r assays are that they r e q u i r e s u p p r e s s i o n o f r e p l i c a t i v e DNA synthesis i f c o n t i n u o u s l y d i v i d i n g l i n e s a r e b e i n g used, and


THE

68


t h a t t h e y a r e d e p e n d e n t upon enzymne p r e p a r a t i o n s for metabolic a c t i v a t i o n . Both of these complicat i o n s are overcome i n the h e p a t o c y t e p r i m a r y c u l ture/DNA r e p a i r assay of Williams (51/52) w h i c h used freshly isolated non-dividing liver cells t h a t can m e t a b o l i z e c a r c i n o g e n s and respond with DNA r e p a i r s y n t h e s i s measured a u t o r a d i o g r a p h i c a l ly. T h i s a s s a y has d e m o n s t r a t e d s u b s t a n t i a l s e n s i t i v i t y and r e l i a b i l i t y w i t h activation-dependent p r o c a r c i n o g e n s (51-53). I t a l s o o f f e r s the advantages of expanded m e t a b o l i c c a p a b i l i t y i n a b a t t e r y and an end p o i n t o f c l e a r b i o l o g i c a l significance. Thus, i t i s a v a l u a b l e a d d i t i o n to b a c t e r i a l mutagenesis assays i n a s c r e e n i n g b a t t e r y . 4.

Chromosome

Tests

Chromosome t e s t s a r e o f c o n c e p t u a l importance b e c a u s e t h e y r e v e a l damage a t a h i g h e r l e v e l of g e n e t i c o r g a n i z a t i o n t h a n do m u t a g e n e s i s assays. T h e r e has been d i f f i c u l t y , however, i n d e v e l o p i n g means o f o b j e c t i v e a n a l y s i s o f many c h r o m o s o m a l alterations. Measurement o f s i s t e r c h r o m a t i d exc h a n g e s ( S C E ) o v e r c o m e s t h i s p r o b l e m and h a s shown s e n s i t i v i t y to c a r c i n o g e n s not r e a d i l y d e t e c t e d i n other i n v i t r o assays (54,5_5). Therefore, determination o f SCEs i s p r e s e n t l y recommended f o r a chromosomal l e v e l test. The resulting extension o f t h e i n f o r m a t i o n b a s e w i t h SCE w i l l be u s e f u l t o delineate further the value and limitations of t h i s r e l a t i v e l y new test. 5.

Cell

Transformation

The f i r s t r e l i a b l e system f o r t r a n s f o r m a t i o n of c u l t u r e d mammalian cells was introduced by S a c h s and a s s o c i a t e s (5j>). T h i s system u t i l i z i n g h a m s t e r f i b r o b l a s t s was s u b s e q u e n t l y d e v e l o p e d i n to a colony assay for quantitative studies by DiPaolo (57) and has been adapted as screening t e s t by P i e n t a e t a l ( 5 8 ) . In a d d i t i o n , a q u a n t i t a t i v e f o c u s a s s a y f o r t r a n s f o r m a t i o n u s i n g mouse cells has been devised in the laboratory of Heidelberger (5j)) and a quantitative assay for g r o w t h o f BHK c e l l s i n s o f t a g a r has been d e v e l oped by Styles (J5 ) . The correlation between t r a n s f o r m a t i o n and m a l i g n a n c y appears t o be g o o d in these systems, but a s u b j e c t of concern is their high frequency of induced transformation. N e v e r t h e l e s s , they p r o v i d e a u s e f u l i n d i c a t i o n o f


6.

WILLIAMS ET AL.

Genetic

Toxicology

in Carcinogen

Testing

the p o t e n t i a l c a r c i n o g e n i c i t y o f c h e m i c a l s either through genotoxic or epigenetic mechanisms and will almost certainly assume a major role in s c r e e n i n g i n the f u t u r e . Another approach under development i s the use of c e l l systems carrying oncogenic v i r u s e s as a more sensitive means of detecting transforming chemicals. Also, because human c a n c e r s u s u a l l y i n v o l v e e p i t h e l i a l tissues, transformation in epithelial systems is actively being pursued. Summary

of

Rapid

In

Vitro

Tests

The five steps (A and B, JL-J5) recommended thus f a r p r o v i d e a b a s i s f o r p r e l i m i n a r y d e c i s i o n making. S u r v e y o f l i t e r a t u r e d a t a on the a p p l i c a t i o n o f t h e recommended t e s t r e v e a l s a h i g h d e g r e e of sensitivity and specificity for this battery (10,28). Evidence of genotoxicity in only one test m u s t be e v a l u a t e d w i t h caution. In particular, several types of chemicals s u c h as intercalating agents are mutagenic to b a c t e r i a , but not r e l i a b l y carcinogenic. A l s o p o s i t i v e r e s u l t s have been obt a i n e d w i t h s y n t h e t i c p h e n o l i c compounds o r n a t u r al products with phenolic structures like flavones. In v i v o , such compounds a r e l i k e l y to be conjugated and excreted readily. Their carcinog e n i c i t y , t h u s , w o u l d d e p e n d o n in v i v o s p l i t t i n g of such c o n j u g a t e s . Therefore, evidence of only b a c t e r i a l m u t a g e n e s i s m u s t be e v a l u a t e d w i t h reg a r d t o the c h e m i c a l s t r u c t u r e and i t s m e t a b o l i s m . I f c l e a r c u t e v i d e n c e o f g e n o t o x i c i t y i n more t h a n one t e s t has been o b t a i n e d , the c h e m i c a l i s highly suspect. Confirmation of carcinogenicity may be sought i n the limited in vivo bioassays without the n e c e s s i t y of resorting to the more c o s t l y and t i m e - c o n s u m i n g c h r o n i c b i o a s s a y . To facilitate the i n t e r p r e t a t i o n of results f r o m a b a t t e r y , B r u s i c k {11) h a s d e v e l o p e d a q u a n t i t a t i v e approach i n which each assay i s a s s i g n e d a n u m e r i c a l v a l u e b a s e d on i t s c o n t r i b u t i o n e i t h e r p o s i t i v e l y o r n e g a t i v e l y t o an a s s e s s m e n t o f g e n o toxicity. The assignment of a value takes into account the f o l l o w i n g : 1. T h e t y p e o f e n d p o i n t m e a s u r e d by t h e t e s t a n d i t s presumed r e l a t i o n s h i p to the development o f c h r o n i c t o x i c i t y ir\ v i v o , i n c l u d i n g m u t a g e n e s i s and c a r c i n o g e n e s i s .



70

2.

The phylogenetic relationship of the test o r g a n i s m t o mammalian s p e c i e s . 3. The reported reproducibility of the test s y s t e m w i t h i n and b e t w e e n l a b o r a t o r i e s . 4. The p u b l i s h e d d a t a base s u p p o r t i n g the utility of the test system to d e t e c t a broad range of chemical c l a s s e s . 5. The s u s c e p t i b i l i t y of the t e s t to incorrect designations of genotoxicity resulting from testing artifacts or anomalous n o n s p e c i f i c responses. 6. The q u a l i t a t i v e similarity between the test system metabolic o r microsome bioactivation s y s t e m and the i n v i v o b i o a c t i v a t i o n mechani s m s i n mammals. 7. The r e s o l v i n g power o f the t e s t system i n cluding the s t r e n g t h of the data analysis methods used w i t h the a s s a y . Applying these criteria, a set of assay v a l u e s was d e v e l o p e d (Table 3). The t e s t s listed i n T a b l e 3 a r e among t h o s e w h i c h h a v e b e e n r o u tinely proposed as s c r e e n i n g methods f o r animal m u t a g e n s and c a r c i n o g e n s and i n c l u d e the b a t t e r y proposed above. Values are assigned f o r p o s i t i v e and negative responses ranging from -5 to +10. The l a r g e s t n e g a t i v e v a l u e r e p r e s e n t s t h e t e s t a n d t e s t c o n d i t i o n s p r o v i d i n g the most p o w e r f u l indication of a lack of genotoxicity. The values between these extremes a r e w e i g h t e d proportionally a c c o r d i n g t o the seven c r i t e r i a l i s t e d above. P o s i t i v e responses are o b v i o u s l y given s i g n i ficantly greater weight than negative results s i n c e n e g a t i v e r e s u l t s c o u l d mean e i t h e r a l a c k o f p o t e n t i a l o r a l a c k o f d e t e c t a b i l i t y by t h e a s s a y . The d i f f e r e n t i a l w e i g h t i n g o f r e s u l t s w i t h o r w i t h o u t an S9 mix i s p r e d i c a t e d upon the assumpt i o n t h a t a s u b s t a n c e a c t i v e w i t h o u t enzyme a c t i vation is unlikely t o show s p e c i e s specificity; w h e r e a s , an a c t i v a t i o n - d e p e n d e n t s u b s t a n c e may be s p e c i e s r e s t r i c t e d and n o t amenable t o g e n e r a l i z e d extrapolation. F o r n e g a t i v e r e s p o n s e s , however, a greater negative value i s assigned to t e s t s employing an activation system. The presence of such a system suggests t h a t n e i t h e r the p a r e n t a l m o l e c u l e nor m i c r o s o m a l l y - p r o d u c e d breakdown p r o d u c t s have d e t e c t a b l e a c t i v i t y . Assignment of the specific values was arbitrary with +10 as a maximum. A f u r t h e r attempt i s made t o b r i n g a c o n s i d eration of potency into the scoring system.


Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981. +2

+3

DNA r e p a i r mammalian

-5

-3

-4

-2

NA

+9

+3

+6

+10

+4

NA

In v i t r o chromosome aberration induction

Sex-linked recessive lethal i n Drosophila

c

S t u d y d e s i g n must i n c l u d e d o s e s e l e c t i o n c r i t e r i a , s u i t a b l e c o n t r o l s , p r o v i s i o n s f o r m u l t i p l e doses. ° Based upon c r i t e r i a and a n a l y s i s c o n s i s t e n t w i t h t h o s e g i v e n i n t h e Appendix. O n l y t e s t s e m p l o y i n g a u t o r a d i o g r a p h i c methods c a n be e v a l u a t e d .

a

Morphological transformation i n v i t r o

induction

-3

SCE

-1

vitro

+2

In

0

and

With Activation

+3

i n cultured cells

0

-4

+7

+8

Ames

Gene m u t a t i o n cultured mammalian c e l l s

Salmonella,

-2

Without Activation

Response

the Weighed

Negative

Using

+5

0

Results

+6

method

Response

Test

With Activation

a

Positive

Score Table f o r Short-term C o n t r i b u t i o n Method.

Procedure

III.

Without Activation

Test

Table

72


Because o f t h e d i v e r s i t y i n end p o i n t s measured, the s p o n t a n e o u s r a t e s f o r the d e t e c t e d e v e n t s and the methods o f s c o r i n g r e s p o n s e s i n the tests, a comparison o f the absolute values i s not f e a s i b l e . A reasonable method to i n c o r p o r a t e potency app e a r s t o be a m e a s u r e m e n t o f t h e l o w e s t t e s t c o n centration producing a biologically significant increaseover the negative control. This value i s designated as the Lowest Positive Concentration Reported (LPCR). Another value d e s i g n a t e d as the Highest Negative Concentration Reported (HNCR) d e f i n e s t h e h i g h e s t t e s t e d c o n c e n t r a t i o n w h i c h was negative. T h e HNCR may be l i m i t e d by t o x i c i t y o r a p r e s e n t maximum a p p l i c a b l e c o n c e n t r a t i o n . The v a l u e s l i s t e d i n T a b l e 3 under P o s i t i v e and Negat i v e r e s p o n s e s may t h e n be m o d u l a t e d by a p o t e n c y f a c t o r a s s e t f o r t h i n T a b l e 4. The p r o d u c t o f t h e t e s t v a l u e s ( T V ) a n d t h e concentration score gives a T o t a l Score (TS) f o r each t e s t o f the b a t t e r y . T h e TS f o r e a c h test will be e i t h e r p o s i t i v e o r n e g a t i v e . The a l g e b r a i c sum r e p r e s e n t s t h e A c t i v i t y S c o r e (AS) f o r t h e compound i n t h e b a t t e r y o f t e s t s t o w h i c h i t has been s u b j e c t e d . The next s t e p i n the s c o r i n g approach i s t o a s s i g n a n e f f e c t d e f i n i t i o n t o t h e AS f o r t h e t e s t substance. The E f f e c t Categories a r e shown i n Table 5 and are calculated i n the f o l l o w i n g manner. 1 A maximum (worst case) g e n o t o x i c e f f e c t , i s c a l c u l a t e d by t a k i n g e a c h a s s a y e m p l o y e d t o evaluate the t e s t m a t e r i a l and calculating the TS products assuming i t was positive without a c t i v a t i o n a t a potency l e v e l equivalent t o t h e maximum tested (not t o exceed 1000 ug/unit). 2. T h i s v a l u e , Maximum P o s i t i v e T o t a l (MP), i s d i v i d e d i n t o t h e AS f o r t h e t e s t m a t e r i a l t o obtain an i n d e x (expressed as p e r c e n t ) of w h a t p o r t i o n o f t h e maximum g e n o t o x i c effect was o b t a i n e d i n t h e e v a l u a t i o n s (% M P ) . 3. The p e r c e n t v a l u e i s then c a t e g o r i z e d u s i n g the E f f e c t T a b l e ( T a b l e 5 ) . 4. Each c a t e g o r y i n the E f f e c t Table d e f i n e s the presumed g e n o t o x i c p o t e n t i a l o f the t e s t subs t a n c e and l e a d s t o an a c t i o n response ( 1 1 ) . Most e x p e r i m e n t a l c a r c i n o g e n s and/or mutagens f a l l i n t o C a t e g o r y 1 (% MP > 6 0 % ) . T h e o n l y p o s s i b l e e x c e p t i o n t o t h i s t r e n d i s benzene which i s not i d e n t i f i e d as a g e n o t o x i c a g e n t by i n v i t r o


6. WILLIAMS ET AL. Table

IV.

Genetic

uG/unit

in Carcinogen

c

a

Converted

Concentration Score Point Factor^

Concentration

Positive Response

b

c

Negative Response

10

1

5

9

1

>6

10

8

1

>11

25

7

1

>26

50

6

1

>51

100

5

2

1.0

-

>101

-

500

4

2

>501

-

1000

3

2

>1001 -

5000

2

2

1

2

>5000 a

73

Testing

Test Concentration Factor f o r ShortTerm T e s t s S c o r e T a b l e U s i n g t h e Weighed C o n t r i b u t i o n Method.

LPCR o r H N C R

to

Toxicology

LPCR = Lowest P o s i t i v e c o n c e n t r a t i o n r e p o r t e d . HNCR = H i g h e s t n e g a t i v e c o n c e n t r a t i o n r e p o r t e d . T h i s f a c t o r i s m u l t i p l i e d by t h e i n d i v i d u a l t e s t score results o b t a i n e d f r o m T a b l e 1. u G / u n i t - C o n c e n t r a t i o n i n micrograms p e r m i l l i l i t e r or per plate, e t c .

Table Percent Maximum P o s i t i v e [MP]

V.

Effect

Table

Classification

> 60%

Potent

Genotoxic

Agent

30 - 5 9 %

Genotoxic

10 - 2 9 %

Suspect

< 10%

I n s u f f i c i e n t Response t o C a t e g o r i z e t h e Agent as Genotoxic

Agent

Genotoxic

Category

1 2

Agent

3 4


74


tests. B e n z e n e has a % MP score of less than 10. Two presumed nongenotoxic carcinogens, s a c c h a r i n and n i t r i l o t r i a c e t i c a c i d , a l s o s c o r e a t a % MP o f l O o r l e s s . B o t h o f t h e s e a g e n t s seem t o represent very little risk as c a r c i n o g e n s Hinder n o r m a l e n v i r o n m e n t a l e x p o s u r e l e v e l s and are not presently c o n t r o l l e d as s i g n f i c a n t human risks. Several compounds such as ascorbate, benzo(e)pyrene and lead acetate fall into c a t e g o r i e s 2 and 3. Compounds i n C a t e g o r y 2, are candidates for limited in vivo bioassays. Chemicals which f a l l i n t o Category 3 are candidates for further genetic evaluation. These include fluorene, diphenylnitrosamine and Fyrol FR-2. T h i s s c o r i n g scheme, t h e n , t a k e s the actual t e s t r e s u l t s from a m u l t i t e s t b a t t e r y through to a s p e c i f i c a c t i o n r e c o m m e n d a t i o n b a s e d on an i n d e x o f t h e maximum g e n o t o x i c e f f e c t f o r t h a t g r o u p o f assays. I t should facilitate the i n t e r p r e t a t i o n and d e c i s i o n m a k i n g p r o c e s s w h i c h f o l l o w t h e a c t u a l t e s t i n g program. If the battery of test systems yields no i n d i c a t i o n o f g e n o t o x i c i t y and thus the chemical falls into category 4 of the action table, the c h e m i c a l may be g i v e n a low p r i o r i t y f o r f u r t h e r t e s t i n g d e p e n d i n g on two c r i t e r i a 1) the struct u r e a n d known p h y s i o l o g i c a l p r o p e r t i e s ( e . g . h o r mone) o f t h e m a t e r i a l and 2) t h e p o t e n t i a l human exposure. I f s u b s t a n t i a l human e x p o s u r e i s l i k e ly, careful consideration should be g i v e n to the necessity for additional testing. The chemical s t r u c t u r e and the p r o p e r t i e s of the m a t e r i a l p r o v i d e d i r e c t g u i d a n c e on t h e p r o p e r r e l e v a n t c o u r s e of a c t i o n . Organic chemicals with structures that present p o s s i b l e s i t e s f o r a c t i v a t i o n may reveal their carcinogenicity in limited in vivo bioassays. On t h e o t h e r h a n d c h e m i c a l s s u c h a s s o l i d state materials, hormones, possibly some metal i o n s and p r o m o t e r s w h i c h a r e n e g a t i v e i n t e s t s f o r g e n o t o x i c i t y o p e r a t e by c o m p l e x and a s y e t poorly understood mechanisms. Thus, i t i s not certain that the limited in vivo bioassays would yield u s e f u l r e s u l t s w i t h such m a t e r i a l s . Therefore the standard chronic bioassay i s , at t h i s time, necess a r y t o d e t e c t any p o t e n t i a l a c t i v i t y with these agents. I t i s indeed urgent to develop reliable means to detect such materials readily without requiring the large investment associated with a chronic bioassay.


6.

WILLIAMS ET AL.

Genetic

Toxicology

in Carcinogen

Testing

The t e s t i n g of metal ions i n rapid bioassay t e s t s may take a d v a n t a g e o f the c o n c e p t recently proposed by L o e b (60) that such ions a f f e c t the f i d e l i t y o f e n z y m e s c o n c e r n e d w i t h DNA synthesis. O b v i o u s l y , the nature of the metal i o n , o f which there are only a limited number, w o u l d provide the n e c e s s a r y i n s i g h t as t o t h e need f o r t e s t i n g such a material further and what k i n d o f assay would most l i k e l y r e v e a l a d v e r s e e f f e c t s . Compounds w i t h h o r m o n e - l i k e properties exist o u t s i d e o f t h e s t r i c t a n d r o g e n and e s t r o g e n t y p e s o f hormones. Such c h e m i c a l s a r e p o t e n t i a l cancer r i s k s m a i n l y because they i n t e r f e r e w i t h the normal physiological endocrine balance (14). More r e s e a r c h o n w a y s a n d means t o t e s t f o r s u c h p r o p erties quickly is required. It i s known, for example, that certain drugs lead to r e l e a s e of p r o l a c t i n from the p i t u i t a r y g l a n d . Chronic i n take of such drugs c a u s i n g permanently higher serum a n d t i s s u e p r o l a c t i n l e v e l s m i g h t i n t u r n a l t e r the r e l a t i v e r a t i o o f o t h e r hormones. With curr e n t u n d e r s t a n d i n g , any m a t e r i a l w i t h s u c h p r o p e r ties needs to undergo a chronic bioassay with carefully and appropriately selected doses to e v a l u a t e whether e n d o c r i n e s e n s i t i v e t i s s u e s would be at higher risk. The interpretation of data m u s t c o n s i d e r t h e n o r m a l d i u r n a l , m o n t h l y and e v e n seasonal cycles of the endocrine system and whether the t e s t would have l e d t o i n t e r f e r e n c e i n this balanced, rythmic system. The potential of halogenated polycyclic h y d r o c a r b o n s t o a c t as p r o m o t e r s i n the p r o d u c t i o n o f l i v e r tumors has been d i s c u s s e d i n d e t a i l ( 1 4 ) . As y e t , t h e s t r u c t u r a l r e q u i r e m e n t s f o r p r o m o t i n g a c t i v i t y are poorly understood o u t s i d e the class o f p h o r b o l e s t e r s , a n d t h e s e a g e n t s c a n be i d e n t i f i e d only i n i n i t i a t i o n - promotion p r o t o c o l s i n l i m i t e d i n vivo bioassays or i n chronic bioassay. The i m p l i c a t i o n s o f the absence o f c o n v i n c i n g data f o r g e n o t o x i c i t y , but a p o s i t i v e response i n chronic bioassays are d i s c u s s e d under the final evaluation. C.

L i m i t e d In V i v o

Bioassays

T h i s stage of e v a l u a t i o n employs t e s t s that w i l l provide further evidence of p o t e n t i a l hazard o f c h e m i c a l s p o s i t i v e f o r g e n o t o x i c i t y w i t h o u t the necessity of undertaking chronic bioassay. A number o f tests for in vivo genotoxicity


76


have been d e v e l o p e d ; these i n c l u d e the dominant lethal test, specific locus test, heritable transl o c a t i o n t e s t , h o s t - m e d i a t e d m u t a g e n i c i t y , chromos o m a l damage, t e s t i c u l a r DNA s y n t h e s i s i n h i b i t i o n , s e b a c e o u s g l a n d s u p p r e s s i o n a n d DNA fragmentation or r e p a i r i n various organs. A chemical that i s negative i n a l l the i n v i t r o g e n o t o x i c i t y tests i s u n l i k e l y t o be p o s i t i v e i n a n y one o f t h e s e i n vivo tests, with p o s s i b l e exception of chemicals a c t i v a t e d t o g e n o t o x i c m e t a b o l i t e s by h o s t b a c t e r ia. Therefore, at present, l i t t l e basis existsfor recommending one o f t h e s e . Furthermore, a positive result i n one o f t h e s e i n v i v o t e s t s w o u l d n o t be c o n c l u s i v e e v i d e n c e o f c a r c i n o g e n i c i t y a n d , t h u s , would s e r v e o n l y as a f u r t h e r i n d i c a t i o n o f the need f o r chronic bioassay, which. as discussed, i s already the only recourse f o r suspect chemicals that are negative i n the i n v i t r o t e s t s . Such i n v i v o tests therefore serve primarily to e s t a b l i s h p r i o r i t i e s f o r c h r o n i c b i o a s s a y o f chemicals negative i n i n v i t r o tests. Thus, a t t h i s s t a g e , the i n v i v o t e s t s recommended a r e t h o s e t h a t w i l l p r o v i d e d e f i n i t i v e e v i dence o f c a r c i n o g e n i c i t y , i n c l u d i n g cocarcinogeni c i t y and p r o m o t i o n , i n a relatively short period (i.e. 30 w e e k s o r l e s s ) . Unlike the i n v i t r o tests, these a r e not a p p l i e d as a b a t t e r y , b u t r a t h e r used s e l e c t i v e l y a c c o r d i n g t o the i n f o r m a t i o n a v a i l a b l e on t h e c h e m i c a l . These t e s t s which have been described in detail by W i l l i a m s and W e i s b u r g e r (28) i n c l u d e 1. S k i n tumor i n d u c t i o n i n m i c e . 2. P u l m o n a r y tumor i n d u c t i o n i n mice 3. Breast cancer i n d u c t i o n i n female Sprague Dawley r a t s 4. A l t e r e d f o c i i n d u c t i o n i n rodent liver E a c h o f t h e s e t e s t s c a n be c o m p l e t e d i n 20-40 weeks and therefore provide a relatively rapid means o f a s s e s s i n g c a r c i n o g e n i c i t y . The c l a s s e s o f c o m p o u n d s a c t i v e i n l i m i t e d i n v i v o b i o a s s a y s a r e shown i n T a b l e 6. L i m i t e d i n v i v o b i o a s a y s a r e recommended f o r substances which y i e l d equivocal results i n the b a t t e r y of s h o r t - t e r m t e s t s o r those p o s i t i v e , but of such major economic s i g n i f i c a n c e that further confirmation i s desired. A l s o , i n the absence o f g e n o t o x i c i t y , i t i s p o s s i b l e to test f o r promoting a c t i v i t y on mouse s k i n i n i t i a t e d w i t h s m a l l d o s e s o f , f o r example, b e n z o ( a ) p y r e n e or 7 ,12-dimethylbenzo(a)anthracene. A material exhibiting


WILLIAMS ET AL. Table VI.

Genetic

Toxicology

in Carcinogen

Carcinogens Active In V i v o B i o a s s a y s

Testing

i n Limited

1.

S k i n Tumors i n Mice A. As Complete C a r c i n o g e n s P o l y c y c l i c a r o m a t i c and h e t e r o c y c l i c hydrocarbons D i r e c t a c t i n g a l k y l a t i n g agents Alkylnitrosoureas B. As I n i t i a t o r s w i t h a P r o m o t e r P o l y c y c l i c aromatic hydrocarbons Certain Arylamines Carbamic a c i d e s t e r s , Urethane C. As P r o m o t e r s o r C o c a r c i n o g e n s with I n i t i a t i o n Phorbol esters Anthralin Catechol

2.

Pulmonary Tumors i n Mice P o l y c y c l i c aromatic hydrocarbons Carbamic a c i d e s t e r s and N - a l k y l a t e d carbamates-urethan Alkylnitrosamides and a l k y l n i t r o s a m i n e s A l k y l a t i n g agents Aziridines Hydrazines Arylamines (poor)

3.

B r e a s t Cancer i n Female Sprague-Dawley P o l y c y c l i c aromatic hydrocarbons Arylamines Alkylnitrosoureas

4.

A l t e r e d F o c i i n Rodent L i v e r A. Rats P o l y c y c l i c aromatic hydrocarbons A r y l a m i n e s , c e r t a i n aminoazo dyes, h e t e r o c y c l i c amines Nitrosamines Urethan Ethionine Aflatoxin Safrole B. Mice Safrole C. Hamsters Nitrosamines

Rats



78

e n d o c r i n e p r o p e r t i e s l i k e w i s e may show an effect in modifying breast cancer induction in animals g i v e n l i m i t e d amounts o f m e t h y l n i t r o s o u r e a as an i n i t i a t i n g dose. S i m i l a r l y , promoters of u r i n a r y bladder cancer may be visulized by pretreatment with limited amounts of a genotoxic bladder carcinogen. Summary

of

limited

in vivo

bioassays

T h e d e t e c t i o n o f two p o s i t i v e r e s u l t s , one i n a b a t t e r y of r a p i d i n v i t r o bioassay t e s t s reli a b l y i n d i c a t i n g g e n o t o x i c i t y and a d e f i n i t e posi tive result i n the limited in vivo bioassays, makes a s u b s t a n c e h i g h l y s u s p e c t as a potential c a r c i n o g e n i c r i s k t o humans. This i s true especi ally i f the r e s u l t s were o b t a i n e d w i t h moderate d o s a g e s and more s o i f t h e r e was evidence of a good dose r e s p o n s e , particularly as r e g a r d s the m u l t i p l i c i t y o f t h e l u n g o r mammary g l a n d t u m o r s . P r o v e n a c t i v i t y i n more t h a n one o f t h e l i m i t e d i n v i v o b i o a s s a y s may be c o n s i d e r e d unequivo c a l q u a l i t a t i v e evidence of c a r c i n o g e n i c i t y . D.

Chronic

Bioassay

Chronic bioassay is used in the decision point approach as a last resort for confirming questionable results i n t h e more l i m i t e d testing o r i n the c a s e o f compounds t h a t a r e n e g a t i v e i n the p r e c e d i n g s t a g e s o f t e s t i n g , but where e x t e n s i v e human e x p o s u r e i s l i k e l y , t h e d e v e l o p m e n t o f d a t a on p o s s i b l e c a r c i n o g e n i c i t y t h r o u g h epigenet i c mechanisms. In the l a t t e r situation, multis p e c i e s and d o s e r e s p o n s e d a t a a r e important. The c o n d u c t o f c h r o n i c b i o a s s a y h a s b e e n d e s c r i b e d i n a number o f r e v i e w a r t i c l e s (61-63). Ε.

Final

Evaluation

If the decision p o i n t approach has led to c h r o n i c b i o a s s a y s , t h e n f a i r l y d e f i n i t i v e d a t a on c a r c i n o g e n i c i t y w o u l d be o b t a i n e d . Nevertheless, the r e s u l t s o f the i n v i t r o s h o r t - t e r m t e s t s are considered f o r e v a l u a t i o n o f p o s s i b l e mechanisms o f a c t i o n a n d r i s k e x t r a p o l a t i o n t o humans. Con vincing positive results i n the in vitro tests coupled with documented in vivo carcinogenicity p e r m i t s c l a s s i f i c a t i o n o f the c h e m i c a l as a geno toxic carcinogen. I t w o u l d , t h e r e f o r e , be a n t i c i -


6.

WILLIAMS ET AL.

Genetic

Toxicology

in Carcinogen

Testing

pated that the chemical could d i s p l a y the propert i e s c h a r a c t e r i s t i c o f such c a r c i n o g e n s which i n clude the a b i l i t y u n d e r some c i r c u m s t a n c e t o be effective as a s i n g l e dose cumulative effects, and synergism or at least additive effects with other genotoxic carcinogens. Genotoxic carcinogens, t h e r e f o r e , r e p r e s e n t c l e a r q u a l i t a t i v e haza r d s t o humans a n d t h e l e v e l o f e x p o s u r e p e r m i t t e d must be rigorously evaluated and controlled. A l o n g t h o s e l i n e s , no d i s t i n c t i o n s h o u l d be made between n a t u r a l l y o c c u r r i n g and s y n t h e t i c c a r c i n o gens. In f a c t , there i s growing evidence that the majority o f human c a n c e r s stem from exposure to the former types o f agents ( 6 4 ) . I f , o n t h e o t h e r h a n d , no c o n v i n c i n g e v i d e n c e f o r g e n o t o x i c i t y i s o b t a i n e d , but the chemical i s c a r c i n o g e n i c i n animal b i o a s s a y s , then the p o s s i b i l i t y e x i s t s that the chemical i s an e p i g e n e t i c carcinogen. The s t r e g n t h o f t h i s c o n c l u s i o n depends upon t h e r e l e v a n c e o f t h e i n v i t r o tests. F o r example, the f i n d i n g that certain stable o r g a n o c h l o r i n e p e s t i c i d e s do n o t d i s p l a y genotoxic e f f e c t s i n l i v e r c e l l systems which a r e i d e n t i c a l to the i n v i v o target c e l l f o r these carcinogens, strongly supports the i n t e r p r e t a t i o n that these c a r c i n o g e n s may a c t by e p i g e n e t i c m e c h a n i s m s . The n a t u r e o f t h e s e mechanisms i s p o o r l y u n d e r s t o o d a t p r e s e n t and i s p r o b a b l y q u i t e d i f f e r e n t f o r d i f ferent classes of carcinogens. T h e y may i n v o l v e chronic tissue injury immunosuppressive effects, hormonal imbalances, blocks in differentiation, promotion of pre-existing altered c e l l s , or processes n o t y e t known. R e g a r d l e s s , most t y p e s o f e p i g e n e t i c carcinogens share the c h a r a c t e r i s t i c o f b e i n g a c t i v e o n l y a t h i g h , s u s t a i n e d d o s e s , a n d up t o a c e r t a i n p o i n t , t h e l e s i o n i n d u c e d may b e r e versible. Thus, these types o f carcinogens may represent only q u a n t i t a t i v e hazards t o humans a n d safe levels of exposure may be e s t a b l i s h e d by carrying out proper pharmacologic dose response studies. Quantitative

Aspects

A number o f d i s t i n c t t y p e s o f c a r c i n o g e n s a n d mutagens d i f f e r i n g i n c h e m i c a l s t r u c t u r e a r e w e l l recognized. These agents d i f f e r from each other as t o e f f e c t i v e n e s s and t a r g e t o r g a n affected i n c a n c e r c a u s a t i o n i n humans a n d i n a n i m a l models. I n many c a s e s , s u c h d i f f e r e n c e s a r e now u n d e r s t o o d


80


as a f u n c t i o n o f b i o c h e m i c a l a c t i v a t i o n l e a d i n g t o the u l t i m a t e c a r c i n o g e n or mutagen,in c o n t r a s t t o detoxification products. For example, benzo(a)pyrene i s a much more p o w e r f u l carcinogen than benzo(a)anthracene, 2-fluoreneamine i s more a c t i v e than 4-biphenylamine, and short chainlength aliphatic nitrosamines are more active than long chainlength compounds. Because of distinct r a t i o s of a c t i v a t i o n over d e t o x i f i c a t i o n metaboli t e s o b t a i n e d i n v i t r o compared t o i n v i v o , t h e s e qualitative and quantitative structure-activity r e l a t i o n s h i p s do n o t a l w a y s h o l d i n s t u d i e s i n volving in vitro effects such as mutagenicity assays. In p a r t i c u l a r , most o f the biochemical a c t i v a t i o n systems used to c o n v e r t promutagens to the a c t i v e m e t a b o l i t e are d e f i c i e n t i n d e t o x i f i c a t i o n a b i l i t y , thus a c c o u n t i n g i n p a r t f o r the l a c k o f c o r r e l a t i o n i n s p e c i f i c i n s t a n c e s (6_5). The primary o b j e c t i v e of mutagenicity and c a r c i n o g e n i c i t y t e s t i n g i s to provide a r e l i a b l e , sound d a t a base f o r r i s k assessment o f e n v i r o n mental chemicals and s i t u a t i o n s with respect to s o m a t i c c e l l e f f e c t s s u c h as n e o p l a s t i c d i s e a s e o r germ c e l l e f f e c t s s u c h as g e n e t i c d i s e a s e s . Thus, t h e t e s t i n g a p p r o a c h d e s c r i b e d s h o u l d be u s e d i n a manner s u c h t h a t d a t a a r e g e n e r a t e d w h i c h c a n i n d e e d be u s e d f o r o b j e c t i v e d e f i n i t i o n o f p o t e n t i a l adverse e f f e c t s . T h i s a s p e c t n e c e s s a r i l y needs to consider q u a n t i t a t i v e potency, i n a d d i t i o n to the q u a l i t a t i v e y e s o r no a n s w e r s . It is quite evident t h a t the p r o t e c t i v e measures needed f o r the liver c a r c i n o g e n a f l a t o x i n Bj^ ( a c t i v e a t 1 ppb) are distinct from those r e q u i r e d f o r the liver c a r c i n o g e n s s a f r o l e ( a c t i v e a t 2000 ppm) or acetamide ( a c t i v e a t 12,500 ppm). The same i s t r u e even f o r the complex e n v i r o n m e n t i n which the general public, as well as specific occupational groups, i s exposed to v a r i e d environmental influe n c e s and h a z a r d s . I t i s beyond the scope o f t h i s r e v i e w d e a l i n g w i t h the r o l e o f g e n e t i c t o x i c o l o g y i n c a r c i n o g e n and m u t a g e n t e s t i n g t o r e v i e w i n d e tail the quantitative aspects of this field. Nevertheless, i t c a n be s t a t e d t h a t t h e c u r r e n t mathematical e v a l u a t i o n s of dose response studies have been based on v e r y few carefully conducted animal bioassays. In fact, a l a r g e number of mathematical models have been based on a single experimental s e r i e s i n v o l v i n g subcutaneous injection of p o l y c y c l i c aromatic hydrocarbons, which because of t h e i r r e l a t i v e insolubility, and slow


6. WILLIAMS ET AL.

Genetic

Toxicology

in Carcinogen

Testing

a b s o r p t i o n f r o m t h e i n j e c t i o n s i t e , e x h i b i t a much broader dose response curve ( a c t i v e o v e r 3-5 l o g u n i t s ) than r a p i d l y absorbed agents such as a r o matic amines o r n i t r o s a m i n e s . F o r example, even with the powerfully carcinogenic N2-fluorenyla c e t a m i d e , a l o w e r i n g o f t h e d o s e by o n l y o n e l o g u n i t , t h a t i s a f a c t o r o f 1 0 , c o n v e r t s a v e r y powe r f u l c a r c i n o g i c s t i m u l u s (200 ppm) t o a v i r t u a l ly inactive dose rate (20 p p m ) . On a larger s c a l e , i n t h e c a s e o f c i g a r e t t e smoke, a n i n d i v i d u a l s m o k i n g 40 s t a n d a r d c i g a r e t t e s p e r d a y h a s a f a i r l y h i g h r i s k o f d i s e a s e , whereas 4 c i g a r e t t e s p e r day w o u l d be a m i n i m a l r i s k . This again i s o n l y a f a c t o r o f 10. Thus, q u a n t i t a t i v e a s p e c t s a r e most i m p o r t a n t i f t h e g o a l o f r i s k e l i m i n a t i o n a n d t h u s d i s e a s e p r e v e n t i o n i s t o be a p p r o a c h e d i n a r e a l i s t i c manner. I n summary, t h e d e c i s i o n p o i n t a p p r o a c h p r o v i d e s a framework f o r s y s t e m a t i c e v a l u a t i o n o f the potential hazards of chemicals, which i n d i c a t e s t h e n e e d f o r a n d c a n be i n t e g r a t e d w i t h o t h e r e l e ments i n t o x i c i t y t e s t i n g ( 6 6 ) . I t i s designed to y i e l d a stepwise progression of data acquisition. An e v a l u a t i o n c a r e f u l l y c o n d u c t e d o f t h i s systema t i c program s h o u l d p r o v i d e s e q u e n t i a l l y a q u a l i tative and a s e m i - q u a n t i t a t i v e d a t a base, which need n o t n e c e s s a r i l y t e r m i n a t e i n an e x p e n s i v e and extensive long-term bioassay, and which provides an e f f e c t i v e t o o l f o r t h e p r o t e c t i o n o f t h e public against environmental cancer and mutagenic risks.

Literature Cited 1.

2. 3.

4.

Bridges, B.A. 1979. Short-term tests and human health—The central role of DNA repair. In Environmental Carcinogenesis, eds. P. Emmelot, E. Kriek, pp. 319-28. Amsterdam: Elsevier/No. Holland Press. Flamm, W.G. 1974. A tier system approach to mutagen testing. Mutat. Res. 26:329-33. Bora, K. C. 1976. A hierarchical approach to mutagenicty testing and regulatory control of environmental chemicals. Mutat. Res. 46:145. Green, S. 1977. Present and future uses of mutagenicity tests for assessment of the safety of food additives. J . Environ. Pathol. Toxicol. 1:49.


82 THE PESTICIDE CHEMIST AND MODERN TOXICOLOGY

5.

6.

7. 8. 9. 10.

11.

12.

13.

14.

Weisburger, J . H . , Williams, G.M. 1978. Decision point approach to carcinogen testing. In Structural Correlates of Carcinogenesis and Mutagenesis, ed. I.M. Asher C. Zervos, pp. 45-52. Rockville, Md. : FDA Office of Health Affairs. Purchase,I.F.H., Longstaff, E.,Ashby, J., Styles,J.A.,Anderson, D., LeFevre, P.A., Westwood,F.R. 1978. An evaluation of 6 short-term tests for detecting organic chemical carcinogens. Br.J. Cancer 37:873-959. Cramer, G.M., Ford, R.A., Hall, R.L. 1978. Estimation of toxic hazard--a decision tree approach. Food Cosmet. Toxicol. 16:255-76. Scientific Committee, Food Safety Council. 1978. Food Cosmet. Toxicol. 16 (Suppl.2):35 Ray, V. 1979. Application of microbial and mammalian cells to the assessment of mutagenicity. Pharmacolo. Revs. 30:537-46. Williams, G . , Kroes, R., van de Poll, K.W. and Waaijers, H.W eds. 1980. The Predictive Value of In Vitro Short-Term Screening Tests in Carcinogenicity Evaluation., Amsterdam: Elsevier/No. Holland Biomed. Press. Brusick, D. Unified scoring system and activity definitions for results from in vitro and submammalian mutagenesis test batteries. In: Proceedings of the Third Life Sciences Symposium on Health Risk Analysis, in press. Hollstein, M., McCann, J., Angelssanto, F.A., Nichols, W.W. 1979. Short-term test for carcinogens and mutagens. Mutat.Res. 46:145. Rinkus, S . J . , Legator, M.S. 1980. The need for both in vitro and in vivo systems in mutagenicity screening. In Chemical Mutagens. Principles and Methods for Their Detection. Vol. 6. de Serres, F.J. and Hollaender, A. pp. 365-473 New York: Plenum Press. Weisburger, J . H . , Williams, G.M. 1980. Chemical carcinogens. In Casarett and Doull's Toxicology, eds. J . Doull C.D. Klaassen, M.O. Amdur, 2nd ed., pp. 84-138. New York. Macmillan.


6. WILLIAMS ET AL.

15.

16. 17.

18.

19. 20.

21.

22.

23.

24.

25. 26.

Genetic Toxicology in Carcinogen Testing

International Commission for Protection Against Environmental Mutagens and Carcinogens 1979. Advice on screening of chemicals for mutagenicity. Mutat. Res. 64:155-58. Privai, M.J. 1979. Genetic toxicology: Regulatory aspects. J . Environ. Pathol. Toxicol. DeSerres, F . J . 1979. Problems associated with the application of short-term tests for mutagenicity in mass-screening programs. Environmental Mutagenesis 1:203-8. Kroes, R. 1979. Animal Data, Interpretation and Consequences. In: Environmental Carcinogenesis: Occurrence, Risk Evaluation and Mechanisms, Emmelot, P., Kriek, E . , eds. pp. 287-299. Amsterdam: Elsevier/No. Holland. Kolbye, A . C . , 1981. The application of fundamentals in risk assessment. American Chemical Society Symposium Series, in press. Mondai, S., Brankow, D.W., and Heidelberger, C. 1978. Enhancement of Oncogenesis in C3H/10T 1/2 mouse embryo cell cultures by saccharin. Science 201:1141-1142. Chang, C.C., Trosko, J . E . and Warren, S.T. 1978. In vitro assay for tumor promoters and antipromoters. J . Environ. Path. & Toxicol. 2:43-64. Weinstein, I.B. 1980. Evaluating substances for promotion, cofactor effects and synergy in the carcinogenic process. J. Environ. Pathol. and Toxicol. 3:89-102. Williams, G.M. 1980. Classification of genotoxic and epigenetic hepatocarcinogens using liver culture assays. Annals New York Academy of Sciences, 349:273-282. Dunkel, V.C. and Williams, G.M. Biological significance of endpoints in short-term tests for carcinogens and mutagens. In: Proceedings of the Third Life Sciences Symposium on Health Risk Analysis, in press. Selkirk, J . 1977. Divergence of metabolic activation systems for short-term mutagenesis assays. Nature 270:604-7. Schmeltz, I., Tosk, J., Williams, G.M. 1978. Comparison of the metabolic profiles of benzo(a)pyrene obtained from primary cell cultures and subcellular fractions derived from normal and methylcholanthrene-induced rat liver. Cancer Letters 5:81-89.


83


27.

28.

29.

30.

31.

32. 33.

34.

35.

36.

Bigger, C.A.H., Tomaszewski, J . E . , Dipple, A. 1980. Limitations of metabolic activa tion systems used with in vitro tests for carcinogens. Science 209:503-4. Williams, G.M. and Weisburger, J.H. System atic carcinogen testing through the decision point approach. Annual Review of Pharmacol ogy and Toxicology, in press. Miller, E.C. and Miller, J.A. 1971. The mutagenicity of chemicals. In Chemical Mutagens: Principles and Methods for Their Detection Vol. 1. A. Hollaender, ed. pp. 83-119 New York: Plenum Press. Weisburger, J.H. and Williams, G.M. 1981. Metabolism of chemical carcinogens. In: Cancer: A Comprehensive Treatise., 2nd Edi tion, Ed. F.F. Becker, Plenum Press, N.Y., pp. 185-234. Ames, B.N. 1979. The identification of chemicals in the environment causing mutations and cancer. In Naturally Occur ring carcinogens-Mutagens and Modulators of Carcinogenesis, eds. E. C. Miller, J.A. Miller, I. Hirono, T. Sugimura, S. Takayama, pp.345-58. Baltimore: University Park Press. Nagao, M., Sugimura, T . , Matsushima, T. 1978. Environmental mutagens and carcino gens. Ann. Rev. Genet. 12:117-59. Rosenkranz, H.S., Poirier, L.A. 1979. Evaluation of the mutagenicity and DNAmodifying activity of carcinogens and noncarcinogens in microbial systems. J . Natl. Cancer Inst. 62:873-92. Chu, Ε.H.Y. Induction and analysis of gene mutations in mammalian cells in culture. In Chemical Mutagens: Principles and Methods for Their Detection, Vol. 1. Hollaender, Α., ed. pp. 411-444. New York: Plenum Press. Radman, M., Caillet-Fauquet, P., Defais, M., Villani, G. 1980.The molecular mechanism of induced mutations and in vitro biochemical assay for mutagenesis. IARC Sci. Pub. 12:537-46. Bartsch, H., Malaveille, C . , Camus, A.M., Martel-Planche, G . , Brun, G . , Hautefeville, Α., Sabadie, Ν., Barbin, Α., Kuroki, T . , Drevon, C . , Piccoli, C. and Montesano, R. Bacterial and mammalian mutagenicity tests:


6. WILLIAMS ET AL. Genetic Toxicology in Carcinogen Testing

37.

38.

39.

40.

41.

42.

validation and comparative studies on 180 Chemicals. In Molecular and Cellular Aspects of Carcinogen Screening Tests. R. Montesano, H. Bartsch and L. Tomatis eds. pp. 179-241. Lyon, France: IARC. Swenberg, J.A. and Petzold, G.L. 1979. The usefulness of DNA damage and repair assays for predicting carcinogenic potential of chemicals. In: Strategies for Short-Term Testing for Mutagens/Carcinogens. B.E. Butterworth, ed. West Palm Beach, Fla.: CRC Press. Painter, R.B. 1978. DNA synthesis inhibi tion in Hela cells as a simple test for agency that damage human DNA. J . Environ. Pathol. & Toxicol. 2:65-78. Stich, H.F., San, R.H.C., Lam, P., Koropatnick and Lo, L. 1977. Unscheduled DNA synthesis of human cells as a short-term assay for chemical carcinogens. In: Ori gins of Human Cancer. Hiatt, H.H., Watson, J.D., and Winston, J.A. eds. pp. 1499-1512 Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory. Williams, G.M. 1979. The status of in vitro test systems utilizing DNA damage and repair for the screening of chemical carcinogens. J. Assoc. Official Analytical Chemists 62:857-63. Ames, B.N., Haroun, L. 1980. An overview of the Salmonella mutagenicity test. In Microsomes, Drug Oxidations, and Chemical Carcinogenesis, vol. 2, eds. M.J. Coon, A.H.Conney R.W. Estabrook, H.V. Gelboin, J.R. Gillette, P.J. O'Brien, pp. 1025-1040. Academic Press. New York. Hyman, J., Leifer, Z . , Rosenkranz, H.S. 1980. The E. coli Pol A assay. A quanti tative procedure for diffusible and nondiffusible chemicals. Mutat. Res. 74:107-11. Sugimura, T., Yahagi, T . , Nagao, Μ., Takeuchi, M., Kawachi, T., Hara, Κ., Yamasaki, E . , Matsushima, T . , Hashimoto, Υ., Okada, M. 1976. Validity of mutagenicity tests using microbes as a rapid screening method for environmental carcinogens. Lyon, France: IARC Sci. Pub. 12:81-104. 1

43.


85


44.

45.

46.

47.

48.

49.

50.

51.

52.

O'Neill, J . P . , Brimer, P.Α., Machanoff, R., Hirsch, G.P., Hsie, A.W. 1977. A quantita tive assay of mutation induction at the hypoxanthine-guanine phosphoribosyl trans ferase locus in Chinese hamster ovary cells (CHO/HGPRT System): Development and defini tion of the system. Mutat. Res. 45:91-101. Huberman, E. 1976. Cell-mediated mutagen icity of different genetic loci in mammalian cells by carcinogenic polycyclic hydrocar bons. In Screening Tests in Chemical Car cinogenesis, eds. R. Montesano, H. Bartsch, L. Tomatis. IARC Sci. Pub. 12, pp.521-36. Lyon, France: IARC. Tong, C . , Williams, G.M. 1980. Definition of conditions for the detection of genotoxic chemicals in the adult rat-liver epithelial cell/hypoxanthine-guanine phosphoribosyl transferase (ARL/HGRPT) mutagenesis assay. Mutat. Res. 74:1-9. Clive, D., Johnson, K.O., Specter, J . F . S . , Batson, A.G., Brown, M.M. 1979. Validation and characterization of the L5178Y/TK mouse lymphoma mutagen assay system. Mutat. Res. 59:61-108. Davies, P . J . , Parry, J . 1974. The induction of oabain-resistant mutants by N-methyl-N'nitro-N-nitrosoguanidine in Chinese hamster cells. Genet. Res. 24:311-14. San, R.H.C., Williams, G.M. 1977. Rat hepatocyte primary cell culture-mediated mutagenesis of adult rat liver epithelial cells by procarcinogens. Proc. Society Experimental Biology Medicine 156:534-38. Roberts, J . J . 1980. Cellular responses to carcinogen-induced DNA damage and the role of DNA repair. British Medical Bulletin, 36:25-31. Williams, G.M. 1980. The detection of chem ical mutagens/carcinogens by DNA repair and mutagenesis in liver cultures In Chemical Mutagens, Principles and Methods for Their Detection vol. 6, eds. F . J . deSerres, A. Hollaender, pp. 61-79. New York Plenum. Williams, G.M. 1980. Liver culture indi cators for the detection of chemical carcin ogens. In: Short-Term Tests for Chemical Carcinogens, eds. R.H.C. San and H.F. Stich, pp. 581-609. New York: Springer-Verlag.


6. WILLIAMS ET AL. Genetic Toxicology in Carcinogen Testing

53.

54. 55.

56. 57.

58.

59. 60. 61.

62. 63.

Probst, G.S., H i l l , L . E . , Brewsey, B.J. 1980. Comparison of three in vitro assays for carcinogen-induced DNA damage. J. Toxi col. Environ. Hlth. 6:333-49. Wolff, S. 1977. Sister chromatid exchange. Annu. Rev. Genet. 11:183-201. Perry, P.E. 1980. Chemical mutagens and sister chromatid exchange. In Chemical Mutagens. Principles and Methods for Their Detection Vol. 6. de Serres, F . J . and Hollaender, A. eds. pp. 1-39 New York: Plenum Press. Berwald, Υ., Sachs, L. 1963. In vitro cell transformation with chemical carcinogens. Nature 200:1182. DiPaolo, J.A. 1979. Quantitative transfor mation by carcinogens of cells in early pas sage. In Environmental Carcinogenesis: Occurrence, Risk Evaluation and Mechanisms, eds., P. Emmelot, E. Kriek, pp. 365-380, Amsterdam: Elsevier/North-Holland. Pienta, R. 1980. Transformation of Syrian hamster embryo cells by diverse chemicals and correlation with their reported carcino genic and mutagenic activities. In Chemical Mutagens: Principles and Methods for their Detection, Vol. 6. de Serres, F . J . and Hollaender A. eds. pp. 175-202. New York: Plenum Press. Heidelberger, C. 1980. Mammalian cell trans formation and mammalian cell mutagenesis. J . Environ. Pathol. Toxicol. 3:69-88. Sirover, M.A. aand Loeb, L.A. 1976. Metalinduced infidelity during DNA synthesis. Proc. Natl. Acad. Sci., 73:2331-2335. Weisburger, J.H. In: Searle, C.E. ed. 1976. Chemical Carcinogenesis. American Chemical Society Monograph 173. New York: American Chemical Society. Sontag, J., ed. 1980. Carcinogens in Indus try and Environment. New York: Marcel Dekker. Page, N.P. 1977. Current concepts of a bio assay program in environmental carcinogene sis. In Advances in Modern Toxicology, vol. 3, ed. H.F. Kraybill, M.A. Mehlman, pp. 87-172. New York: John Wiley.

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