Role of StructureActivity Relationship Analysis in Evaluation of

Chapter 11

Carcinogenicity and Pesticides Downloaded from pubs.acs.org by MONASH UNIV on 11/11/15. For personal use only.

Role of Structure-Activity Relationship Analysis in Evaluation of Pesticides for Potential Carcinogenicity Yin-tak Woo and Joseph C. Arcos Office of Toxic Substances, U.S. Environmental Protection Agency, Washington, DC 20460

Structure-activity relationship (SAR) analysis i s essential for the development of pesticides and for the evaluation of cancer hazard and r i s k assessment. The c r i t i c a l factors that should be considered in SAR analysis and the p r o f i l e of typical potent carcinogens are discussed. A scheme combining structural and functional c r i t e r i a for suspecting chemical compounds of carcinogenic activity is presented. Selected classes of pesticides with carcinogenic potential are reviewed to exemplify structural and/or functional features responsible for their carcinogenic activity. Structure-activity relationship (SAR) analysis is a critical tool in the research and development of new industrial and agricultural chemicals and is the f i r s t line of approach in the cancer hazard evaluation of chemicals. Careful SAR analyses can spot or reveal potential health hazard of new chemicals early in the research and development stage. SAR considerations are also essential for designing and selecting appropriate batteries of tests to study the potential toxicity of chemicals and to elucidate their molecular mechanisms of action. There are various ways to approach SAR analysis. This chapter focuses on principles and concepts of mechanism-based SAR analysis along with an overview of the structural features and critical factors that should be considered in the evaluation of pesticides for potential carcinogenicity. Most of principles and This chapter not subject to U.S. copyright Published 1989 American Chemical Society

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concepts o f SAR analysis covered i n this chapter r e p r e s e n t a d i s t i l l a t e b a s e d on a s e r i e s o f monographs (1-5) a n d r e v i e w s (6,7) on t h e c h e m i c a l i n d u c t i o n o f c a n c e r by t h e a u t h o r s . Readers a r e r e f e r r e d t o these s o u r c e s ( p a r t i c u l a r l y t h e C u m u l a t i v e I n d e x i n r e f . 5) f o r t h e o r i g i n a l r e f e r e n c e s o f s p e c i f i c SAR s t u d i e s .


Multi-stage and Carcinogenesis

Multifactorial

Nature

of

Chemical

The SAR analysis of carcinogens requires a basic u n d e r s t a n d i n g o f t h e i r b i o c h e m i c a l mechanisms o f a c t i o n . C h e m i c a l c a r c i n o g e n e s i s may h a v e many e t i o l o g i c a l f a c t o r s and i s a c o m p l e x p r o c e s s t h a t may be d i v i d e d i n t o a t least three d i s t i n c t stages: i n i t i a t i o n (DNA damage which, i f u n r e p a i r e d o r m i s r e p a i r e d , e v e n t u a l l y l e a d s t o t h e f o r m a t i o n o f p r e n e o p l a s t i c o r "dormant" tumor c e l l s ) , promotion ( i n which p r e n e o p l a s t i c c e l l s a r e expressed i n t o i n d i v i d u a l tumor c e l l s ) a n d p r o g r e s s i o n ( i n v o l v i n g p r o g r e s s t o malignancy by h i s t o p a t h o l o g i c c r i t e r i a ) . A v a r i e t y o f endogenous ( h o s t ) f a c t o r s s u c h a s immune competence, hormonal r e g u l a t i o n , and exogenous f a c t o r s such as d i e t , r a d i a t i o n , trauma/stress can p l a y important c o n t r i b u t o r y r o l e s . Chemical carcinogens can e x e r t t h e i r a c t i o n by d i r e c t l y a c t i n g on t h e s e t h r e e s t a g e s o f carcinogenesis as w e l l as indirectly through the endogenous (host) f a c t o r s .

Mechanistic C l a s s i f i c a t i o n

o f Chemical

Carcinogens

From the point o f view o f mechanism of action, c a r c i n o g e n s c a n be l o o s e l y c l a s s i f i e d a s : ( i ) g e n o t o x i c c a r c i n o g e n s , and ( i i ) e p i g e n e t i c c a r c i n o g e n s . G e n o t o x i c c a r c i n o g e n s c a u s e DNA damage d i r e c t l y , m o s t l y through c o v a l e n t b i n d i n g t o DNA, a n d a r e a l s o c a l l e d D N A - r e a c t i v e c a r c i n o g e n s . D e s p i t e t h e i r s t r u c t u r a l v a r i e t y , t h e y have one f e a t u r e i n common — they are e i t h e r e l e c t r o p h i l e s p e r s e o r c a n be a c t i v a t e d t o e l e c t r o p h i l i c reactive intermediates. Epigenetic carcinogens do not bind c o v a l e n t l y t o DNA, do n o t c a u s e DNA damage d i r e c t l y , a n d u s u a l l y produce negative o r i n c o n s i s t e n t r e s u l t s i n s h o r t - t e r m t e s t s f o r g e n o t o x i c i t y . They a c t by a v a r i e t y o f n o t c l e a r l y d e f i n e d e x t r a c h r o m o s o m a l mechanisms s u c h as peroxisome p r o l i f e r a t i o n , i n h i b i t i o n o f i n t e r c e l l u l a r communication, hormonal imbalance, cytotoxicity, etc. P r e d i c t i o n o f t h e i r p o s s i b l e mechanism o f a c t i o n i s i m p o r t a n t f o r m e a n i n g f u l SAR c o n s i d e r a t i o n s . Carcinogens which a c t as e l e c t r o p h i l i c r e a c t a n t s may be further classified as: (i) direct-acting carcinogens which are reactive as such, and ( i i ) indirect-acting carcinogens which r e q u i r e activation

11. WOO&ARCOS

Evaluation ofPesticides for Potential Carcinogenicity

chemically (e.g., by a c i d o r base), photochemically ( e . g . , uv) o r m e t a b o l i c a l l y . I n g e n e r a l , d i r e c t - a c t i n g c a r c i n o g e n s t e n d t o be l o c a l l y a c t i v e whereas t h o s e which r e q u i r e a c t i v a t i o n a r e u s u a l l y c a r c i n o g e n i c mostly toward t i s s u e ( s ) where a c t i v a t i o n o c c u r s .


Critical

F a c t o r s f o r SAR C o n s i d e r a t i o n

There are four critical features that should be c o n s i d e r e d i n SAR a n a l y s i s : ( i ) p h y s i c o c h e m i c a l , ( i i ) molecular geometric, ( i i i ) metabolic, and ( i v ) mechanistic. Physicochemical factors: Irrespective of i t s chemical structure o r mechanism of action, the carcinogenic potential of a chemical compound i s dependent on i t s p h y s i c o c h e m i c a l properties which determine i t s " b i o a v a i l a b i l i t y " , t h a t i s , i t s a b i l i t y t o r e a c h t a r g e t t i s s u e s a n d c e l l s . T h e most s a l i e n t o f t h e s e properties are: (1) Molecular weight: Compounds with very high m o l e c u l a r w e i g h t ( o v e r 1,000-1,500) a n d s i z e h a v e l i t t l e chance o f b e i n g absorbed i n s i g n i f i c a n t amounts; i n g e n e r a l , t h e y do n o t p o s e a n y s u b s t a n t i a l c a r c i n o g e n i c r i s k . There a r e o f course important exceptions t o t h e r u l e . H i g h M.W. compounds w h i c h c a n be d e g r a d e d i n t h e g a s t r o i n t e s t i n a l t r a c t (e.g., by h y d r o l y s i s o r m i c r o b i a l a c t i o n ) s h o u l d be a s s e s s e d c o n s i d e r i n g t h e i r p r o b a b l e degradation products. Certain high M.W. polymeric substances (e.g., degraded carageenan) have local c a r c i n o g e n i c e f f e c t i f i n g e s t e d , i n h a l e d o r i n j e c t e d (see ref. 5). (2) P h y s i c a l s t a t e : The p h y s i c a l s t a t e o f a c h e m i c a l compound may, t o some e x t e n t , a f f e c t i t s c a p a b i l i t y t o reach target tissues. Compounds which are highly v o l a t i l e , o r w h i c h c a n b e i n h a l e d a s d u s t p a r t i c l e s , may have " d i r e c t a c c e s s " t o nasopharyngeal and/or pulmonary tissues. (3) S o l u b i l i t y : I n g e n e r a l , compounds w h i c h a r e h i g h l y h y d r o p h i l i c a r e p o o r l y absorbed and, i f absorbed, a r e r e a d i l y e x c r e t e d . Thus, t h e i n t r o d u c t i o n o f h y d r o p h i l i c groups (e.g., s u l f o n y l , carboxyl) i n t o an o t h e r w i s e c a r c i n o g e n i c compound u s u a l l y m i t i g a t e s a n d s o m e t i m e s altogether abolishes i t s carcinogenic activity. (4) Chemical reactivity: Although many chemical compounds owe t h e i r carcinogenic activity to their e l e c t r o p h i l i c c h e m i c a l r e a c t i v i t y , compounds w h i c h a r e "too reactive" a r e n o t c a r c i n o g e n i c . Compounds a r e considered "too r e a c t i v e " i f they hydrolyze o r polymerize spontaneously and i n s t a n t a n e o u s l y , or react with n o n c r i t i c a l c e l l u l a r c o n s t i t u e n t s before they can reach t a r g e t t i s s u e s and r e a c t w i t h key macromolecules. F o r example, the carcinogenic activity of the reactive

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electrc-phile, β - p r o p i o l a c t o n e c a n be a b o l i s h e d by t h e i n t r o d u c t i o n o f an e x o c y c l i c d o u b l e bond w h i c h makes t h e compound ( d i k e t e n e ) " t o o r e a c t i v e " ( s e e r e f . 3 ) . I t i s important t o p o i n t out t h a t the route of exposure i s a k e y f a c t o r i n c o n s i d e r i n g w h e t h e r t h e compound i s " t o o reactive". F o r example, b i s - c h l o r o m e t h y l e t h e r may be considered "too r e a c t i v e " i f administered o r a l l y in a q u e o u s s o l u t i o n ( t a p p r o x i m a t e l y 40 s e c o n d s ) , however, t h e compound i s a p o t e n t n a s a l / p u l m o n a r y carcinogen i f i n h a l e d as v a p o r ( t i n humid a i r may be a s l o n g a s 25 hours) (see r e f . 3 ) . 1 / 2


1 / 2

Molecular geometric factor: Molecular size and g e o m e t r y o f a c h e m i c a l compound a f f e c t i t s a b i l i t y t o r e a c h t a r g e t t i s s u e and t a r g e t m a c r o m o l e c u l e s and i t s c h a n c e t o be m e t a b o l i c a l l y a c t i v a t e d o r d e t o x i f i e d . Many p o t e n t c a r c i n o g e n s / m u t a g e n s h a v e a common f e a t u r e — they have a molecular size and shape favorable for i n t e r c a l a t i o n i n t o DNA p l u s a r e a c t i v e o r p o t e n t i a l l y r e a c t i v e f u n c t i o n a l g r o u p . F o r example, i t h a s been c a l c u l a t e d ( s e e réf. 1) t h a t most p o t e n t c a r c i n o g e n i c p o l y c y c l i c a r o m a t i c hydrocarbons have a p l a n a r s t r u c t u r e with 4 - 6 r i n g s ( o f w h i c h n o t more t h a n f o u r may be l i n e a r l y c o n n e c t e d ) and a m o l e c u l a r s i z e o f b e t w e e n a b o u t 90 t o 180 A . T h i s s i z e r e q u i r e m e n t coupled with the p o t e n t i a l t o be a c t i v a t e d t o e l e c t r o p h i l i c bay-region d i o l e p o x i d e r e p r e s e n t two c r i t i c a l f a c t o r s t h a t d e t e r m i n e the carcinogenic activity of polycyclic aromatic h y d r o c a r b o n s . The p o t e n t c a r c i n o g e n s f o u n d i n o v e r c o o k e d f o o d ( e . g . . T r p - P - 1 , P-2 ; G l u - P - 1 , P-2) a r e m o s t l y p l a n a r t r i c y l c i c compounds (8) w i t h amino g r o u p ( s ) t h a t c a n be activated to e l e c t r o p h i l i c nitrenium ions. Many o t h e r p o t e n t c a r c i n o g e n s and mutagens (e.g., a f l a t o x i n B psoralen-8-glycidyl ether, acridine mustard, 2acetylaminofluorene) are planar molecules with a favorable molecular size bearing an electrophilic f u n c t i o n a l group (see r e f s . 2-5). 2

w

Metabolic factor: Metabolism can both a c t i v a t e and d e t o x i f y chemical carcinogens. For direct-acting carcinogens, metabolism tends t o decrease the a c t i v i t y . Many p e s t i c i d e s a r e d i r e c t - a c t i n g e l e c t r o p h i l e s . T h u s , an e s t i m a t e o f t h e e x t e n t o f m e t a b o l i c d e t o x i f i c a t i o n o f t h e p e s t i c i d e s i s e s s e n t i a l f o r a c c u r a t e SAR p r e d i c t i o n . For indirect-acting carcinogens, a delicate balance between the activation and detoxification pathways d e t e r m i n e s t h e c a r c i n o g e n i c i t y o f t h e compound. Knowledge of the metabolic pathways of these chemicals can s u b s t a n t i a l l y e n h a n c e t h e a c c u r a c y o f SAR a n a l y s i s . F o r example, for carcinogenic polycyclic aromatic hydrocarbons, i n t r o d u c t i o n o f s m a l l s u b s t i t u e n t s (such as methyl o r f l u o r o ) a t t h e L - r e g i o n i n c r e a s e s t h e c a r c i n o g e n i c i t y , w h e r e a s any s u b s t i t u t i o n a t t h e s i t e o f activation (the b a y - r e g i o n benzo r i n g ) d e c r e a s e s or a n n u l s c a r c i n o g e n i c i t y ( s e e r e f s . 1, 9, 1 5 ) .


11. WOO&ARCOS


A v a r i e t y o f o x i d a t i v e , r e d u c t i v e , h y d r o l y t i c and c o n j u g a t i n g enzymes/enzyme s y s t e m s a c t i v a t e chemical c a r c i n o g e n s ( 7 ) . The m i x e d - f u n c t i o n o x i d a s e s a r e by f a r the most well known activating system f o r most carcinogens ( 1 0 ) . However, at least two other enzymes/enzyme s y s t e m s d e s e r v e a s p e c i a l m e n t i o n h e r e . One i s the relatively little known p r o s t a g l a n d i n H s y n t h a s e (PHS) t h a t i s i n c r e a s i n g l y c o n s i d e r e d t o b e t h e m a j o r a c t i v a t i n g s y s t e m i n many e x t r a h e p a t i c t i s s u e s (11) . T h e PHS s y s t e m h a s b e e n shown t o a c t i v a t e a v a r i e t y of chemical carcinogens including some that were p r e v i o u s l y assumed t o b e " e p i g e n e t i c " ( s e e r e f . 7 ) . T h e herbicide, amitrole (3-amino-l,2,4-triazole), a thyroid c a r c i n o g e n and g o i t r o g e n , f o r example, h a s b e e n g e n e r a l l y assumed t o b e a n " e p i g e n e t i c " c a r c i n o g e n b e c a u s e o f i t s g o i t r o g e n i c a c t i v i t y and i t s i n a c t i v i t y i n a v a r i e t y o f short-term mutagenicity assays (mostly using liver m i c r o s o m e s a s t h e a c t i v a t i n g enzyme s o u r c e ) . However, r e c e n t s t u d i e s i n d i c a t e t h a t a m i t r o l e c a n i n d u c e gene mutation and i n v i t r o c e l l t r a n s f o r m a t i o n i n S y r i a n h a m s t e r embryo f i b r o b l a s t s w h i c h a r e known t o c o n t a i n PHS a c t i v i t y . A m i t r o l e c a n be a c t i v a t e d by microsomes from ram s e m i n a l v e s i c l e s ( h i g h i n PHS a c t i v i t y ) t o r e a c t i v e i n t e r m e d i a t e s c a p a b l e o f b i n d i n g c o v a l e n t l y t o DNA ( 1 2 a ) . Another interesting enzyme i s g l u t a t h i o n e (GSH) Stransferase which i s g e n e r a l l y regarded t o be a d e t o x i f y i n g enzyme. However, f o r d i h a l o a l k a n e s s u c h a s t h e f u m i g a n t , 1 , 2 - d i b r o m o e t h a n e , GSH S - t r a n s f e r a s e i s a n a c t i v a t i n g r a t h e r t h a n a d e t o x i f y i n g enzyme b e c a u s e t h e m e t a b o l i t e f o r m e d , a GSH c o n j u g a t e , i s a c t u a l l y a h a l f sulfur mustard (GS-CH -CH -C1) which can undergo cyclization to yield electrophilic episulfonium i n t e r m e d i a t e ( s e e r e f s . 7, 1 2 b ) . 2

2

Mechanistic factor: D e p e n d i n g on t h e p r o j e c t e d mechanism o f a c t i o n , d i f f e r e n t a p p r o a c h e s a r e n e e d e d i n SAR a n a l y s i s . However, s i n c e c a r c i n o g e n s o f t e n a c t b y more t h a n one mechanism, a l l p o s s i b l e mechanisms s h o u l d be c o n s i d e r e d f o r e v a l u a t i o n o f t h e i r a p p l i c a b i l i t y t o the chemical i n question.

Profile

of Typical

Potent

Carcinogens

T h e r e a r e a number o f c h a r a c t e r i s t i c s t h a t a r e common t o most t y p i c a l p o t e n t c a r c i n o g e n s : (1) F i r s t , t h e y must b e a b l e t o r e a c h t a r g e t s i t e s a t sufficiently high level. As d i s c u s s e d e a r l i e r , t h e physicochemical p r o p e r t i e s o f t h e chemical play a determining r o l e . Reactive d i r e c t - a c t i n g e l e c t r o p h i l i c chemicals are often potent carcinogens i f they are e i t h e r

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(i) volatile (e.g., bis-chloromethyl ether), ( i i ) a d m i n i s t e r e d by r o u t e s o r c o n d i t i o n s a l l o w i n g d i r e c t access t o t a r g e t tissues, or ( i i i ) r e s i s t a n t t o metabolic detoxification (e.g., 1,2-dibromoethane which is a c t i v a t e d r a t h e r t h a n d e t o x i f i e d b y GSH). (2) A c t i v a t i o n n e a r o r a t t h e s i t e o f t a r g e t i s one o f the most effective means of generating potent carcinogens. For this reason, the l i v e r , t h e most i m p o r t a n t o r g a n f o r m e t a b o l i s m , i s a most f r e q u e n t l y observed t a r g e t organ f o r carcinogens that require m e t a b o l i c a c t i v a t i o n . A number o f c a r c i n o g e n s ( e . g . , 4aminobiphenyl) can be activated to electrophilic i n t e r m e d i a t e s i n t h e l i v e r , t e m p o r a r i l y d e t o x i f i e d by forming conjugates (e.g., g l u c u r o n i d e ) , t r a n p o r t e d i n t h i s " p r o t e c t e d " form t o a second t a r g e t (e.g., u r i n a r y b l a d d e r ) , and r e a c t i v a t e d by t a r g e t - s p e c i f i c h y d r o l a s e (e.g., β-glucuronidase) o r a c i d h y d r o l y s i s (see r e f s . 2, 7, 1 2 b ) . (3) A r e a s o n a b l e l i f e t i m e t o p e r m i t i n t e r a c t i o n w i t h t a r g e t macromolecules i s a l s o c r u c i a l . For reactive carcinogens, t h i s means some ways t o s t a b i l i z e t h e reactive intermediate (see d i s c u s s i o n under "SAR C o n s i d e r a t i o n o f Genotoxic Carcinogens") . F o r n o n r e a c t i v e c a r c i n o g e n s , t h i s means m e t a b o l i c s t a b i l i t y s o t h a t t h e c h e m i c a l may s t a y i n t h e body l o n g e r . (4) S e l e c t i v e , s p e c i f i c , and p e r s i s t e n t interaction with DNA o r o t h e r t a r g e t m a c r o m o l e c u l e s i s a n o t h e r c r i t i c a l f e a t u r e . F o r example, t h e p o t e n t c a r c i n o g e n , aflatoxin B after metabolic activation binds s e l e c t i v e l y t o c e r t a i n s p e c i f i c r e g i o n s o f DNA. The DNA a d d u c t s t h u s formed undergo p o s t - b i n d i n g c o n v e r s i o n t o a d d u c t s t h a t a r e p e r s i s t e n t and r e s i s t a n t t o DNA r e p a i r enzymes ( s e e r e f . 5) . O t h e r p o t e n t c a r c i n o g e n s s u c h a s 7,12-dimethylbenz[a]anthracene and N - n i t r o s o m e t h y l u r e a h a v e b e e n shown t o s e l e c t i v e l y b i n d t o s p e c i f i c DNA r e g i o n s t h a t e n c o d e o n c o g e n e s ( 1 3 ) . F o r 2 , 3 , 7,8-TCDD, i t s e l e c t i v e l y b i n d s t o c y t o s o l i c Ah p r o t e i n . A l t h o u g h t h e binding i s not covalent, the persistent nature of 2,3,7,8-TCDD ensures mechanistically significant i n t e r a c t i o n (14). (5) A s d i s c u s s e d e a r l i e r , c h e m i c a l c a r c i n o g e n e s i s i s a c o m p l e x p r o c e s s i n v o l v i n g m u l t i p l e s t a g e s a n d many f a c t o r s . Most p o t e n t c h e m i c a l c a r c i n o g e n s e x e r t e f f e c t s on m u l t i p l e s t e p s o r s t a g e s o f c a r c i n o g e n e s i s . The synergistic o r complementary combination of these individual effects determines t h e potency of the carcinogen. For example, the potent carcinogen, benzo[a]pyrene, i s metabolically activated to reactive b a y - r e g i o n d i o l e p o x i d e t h a t b i n d s t o DNA a n d i n i t i a t e s carcinogenesis (15), i t i s a l s o metabolized t o phenolic d e r i v a t i v e s t h a t a c t as promotors (16), and i t i s a c t i v e a s a n immune s u p p r e s s i v e a g e n t ( 1 7 ) . w

11. WOO&ARCOS SAR


Consideration o f Genotoxic

Carcinogens


As d i s c u s s e d above, SAR a n a l y s i s i s more e f f e c t i v e i f we c a n p r e d i c t / p r o j e c t t h e p o s s i b l e mechanism o f a c t i o n o f t h e c h e m i c a l . F o r g e n o t o x i c c a r c i n o g e n s , SAR a n a l y s i s should include c o n s i d e r a t i o n of: (i) t h e nature o f t h e e l e c t r o p h i l e p r e s e n t o r t o be formed, ( i i ) p o s s i b i l i t y of s t a b i l i z a t i o n o f t h e i n t e r m e d i a t e , and ( i i i ) t h e molecule t o which t h e e l e c t r o p h i l e i s attached. The electrophiles or electrophilic intermediates t h a t a r e o r a r e p o s t u l a t e d t o be r e s p o n s i b l e f o r t h e carcinogenic a c t i o n o f chemicals include: (i) p o s i t i v e l y c h a r g e d c a r b o n i u m , n i t r e n i u m , oxonium a n d e p i s u l f o n i u m i o n s , ( i i ) f r e e r a d i c a l s , ( i i i ) p o l a r i z e d double bonds, (iv) aldehydes, (v) s t r a i n e d r i n g s s u c h a s e p o x i d e , aziridine, l a c t o n e s and s u l t o n e s , and ( v i ) quinone/ quinoid/quinoneimine structures. Based on their r e a c t i v i t y ( T a b l e I) , e l e c t r o p h i l e s may b e g r a d e d f r o m " s o f t " t o "hard" s i m i l a r t o t h e concept o f " s o f t " and "hard" acids and bases (18). In general, soft electrophiles react p r e f e r e n t i a l l y with soft nucleop h i l e s whereas h a r d e l e c t r o p h i l e s r e a c t p r e f e r e n t i a l l y w i t h h a r d n u c l e o p h i l e s . Thus, s i n c e t h e n u c l e o p h i l i c s i t e s i n t h e p u r i n e a n d p y r i m i d i n e b a s e s i n DNA a r e moderately hard nucleophiles, moderately hard e l e c t r o p h i l e s t e n d t o have t h e g r e a t e s t l i k e l i h o o d o f c o v a l e n t b i n d i n g t o DNA. S o f t e l e c t r o p h i l e s o f t e n d e p l e t e t h e c e l l u l a r p o o l o f n o n c r i t i c a l s o f t n u c l e o p h i l e s (such a s GSH) b e f o r e t h e y c a n r e a c t w i t h DNA. E v e n when a c t i v a t e d n e a r t h e t a r g e t s i t e , some e l e c t r o p h i l i c intermediates a r e s t i l l too r e a c t i v e t o t r a v e l f r o m e n z y m i c s i t e t o DNA. R e s o n a n c e s t a b i l i z a t i o n o f t e n p r o v i d e s t h e needed l e n g t h e n i n g o f l i f e t i m e f o r t h e r e a c t i v e intermediate t o reach i t s t a r g e t macromolecules. A c o m p r e h e n s i v e s t u d y o f t h e SAR o f c a r c i n o g e n i c a r o m a t i c a m i n e s (2) b e s t i l l u s t r a t e s t h e p o i n t . F i g u r e 1 shows t h e position(s) where a t t a c h m e n t o f a n amine o r a m i n e g e n e r a t i n g group (such a s n i t r o , n i t r o s o group) can y i e l d c a r c i n o g e n i c a r o m a t i c amines. These p o s i t i o n s c o r r e s p o n d t o t h e t e r m i n a l end(s) o f t h e l o n g e s t conjugated system i n t h e molecule. I t i s a t these p o s i t i o n s that t h e reactive nitrenium ions, generated by metabolic activation, c a n b e most effectively stabilized by resonance w i t h t h e aromatic r i n g system. The molecular segment a d j a c e n t / a t t a c h e d t o the e l e c t r o p h i l i c r e a c t i v e s i t e / g r o u p can a f f e c t c a r c i n o g e n i c p o t e n t i a l i n a v a r i e t y o f ways. I n a d d i t i o n t o m o l e c u l a r s i z e and resonance s t a b i l i z a t i o n , a number o f o t h e r molecular parameters c a n modulate the carcinogenic p o t e n t i a l o f t h e e l e c t r o p h i l i c moiety. Attachment o f normal c e l l u l a r c o n s t i t u e n t s o r t h e i r s t r u c t u r a l analogs

181

182


TABLE I


T y p i c a l " S o f t " and " H a r d " E l e c t r o p h i l e s and T h e i r P r e f e r r e d S i t e s o f A t t a c k i n Macromolecules

Examples o f S o f t Soft

and H a r d

Electrophiles

Aldehydes, p o l a r i z e d

d o u b l e bonds

Epoxides, s t r a i n e d - r i n g lactones, alkyl sulfates, alkyl halides Arylcarbonium Benzylic ions Hard

carbonium i o n s ,

Alkylcarbonium Nucleophilic

Soft

Sites

ions nitrenium

ions

i n Macromolecules

T h i o l groups o f c y s t e i n y l r e s i d u e s i n p r o t e i n and g l u t a t h i o n e S u l f u r atoms o f m e t h i o n y l in protein

residues

P r i m a r y amino g r o u p s o f a r g i n i n e l y s i n e residues i n protein

and

Amino g r o u p s o f p u r i n e and p y r i m i d i n e b a s e s i n RNA and DNA Oxygen atoms o f p u r i n e and p y r i m i d i n e b a s e s i n RNA and DNA Hard

1

Brian

Coles,

P h o s p h o r y l o x y g e n atoms i n b a c k b o n e o f RNA and DNA

personal

communication.



11. WOO&ARCOS

Fig. 1. Role o f Resonance Stabilization in C o n t r i b u t i n g t o C a r c i n o g e n i c A c t i v i t y o f A r o m a t i c Amines. The l o w e r d i a g r a m i n d i c a t e s t y p i c a l h y d r o c a r b o n m o i e t i e s p r e s e n t i n c a r c i n o g e n i c a r o m a t i c amines. The unconnected b o n d ( s ) i n t h e s e m o i e t i e s i n d i c a t e t h e p o s i t i o n where a t t a c h m e n t o f (an) amine o r a m i n e - g e n e r a t i n g g r o u p ( s ) y i e l d s c a r c i n o g e n i c compounds. T h e s e p o s i t i o n s c o r r e s p o n d t o t h e t e r m i n a l end(s) o f l o n g e s t c o n j u g a t e d system i n t h e m o l e c u l e a n d a r e t h e most f a v o r a b l e p o s i t i o n s f o r resonance stabilization of reactive nitrenium ions g e n e r a t e d by m e t a b o l i c a c t i v a t i o n (see upper b l o c k e d diagram).

183



184

to the electrophilic moiety can often increase c a r c i n o g e n i c i t y . I n t h e s e i n s t a n c e s t h e m o l e c u l a r segment a t t a c h e d a c t s as a c a r r i e r t o f a c i l i t a t e e n t r y i n t o t h e t a r g e t o r g a n ( s ) . F o r example, t h e p a n c r e a t i c c a r c i n o g e n , s t r e p t o z o t o c i n , c o n t a i n s a N - n i t r o s o m o i e t y and a s u g a r m o i e t y . The f o r m e r g e n e r a t e s an e l e c t r o p h i l i c m o i e t y whereas t h e l a t t e r a c t s as a c a r r i e r ( see r e f . 5) . A t t a c h i n g the r e a c t i v e mustard group t o u r a c i l y i e l d s a h i g h l y p o t e n t c a r c i n o g e n ( s e e r e f . 3) , p r e s u m a b l y b e c a u s e u r a c i l c a n be r e a d i l y t a k e n up by c e l l s . The l o c a t i o n o f the e l e c t r o p h i l i c moiety(ies) i n the molecule i s a l s o important. E l e c t r o p h i l i c moiety(ies) situated at the t e r m i n a l ends h a s ( v e ) a b e t t e r chance o f r e a c t i n g w i t h target macromolecules than those situated at or s t e r i c a l l y " b u r i e d " i n the middle of the molecule because o f s t e r i c h i n d r a n c e . M o l e c u l e s c o n t a i n i n g more t h a n one e l e c t r o p h i l i c m o i e t y a r e more l i k e l y t o be c a r c i n o g e n i c than monofunctional ones particularly i f the e l e c t r o p h i l i c m o i e t i e s a r e f r e e l y f l e x i b l e and n o t t o o f a r a p a r t ( s e e r e f . 3) .

SAR

Consideration

of Epigenetic

Carcinogens

In contrast to genotoxic carcinogens, epigenetic c a r c i n o g e n s a c t by a w i d e v a r i e t y o f mechanisms. Some o f the possible epigenetic mechanisms of chemical carcinogenesis include: peroxisome proliferation, i n h i b i t i o n o f i n t e r c e l l u l a r communication, microtubule alteration, hormonal imbalance, cytotoxicity, immunomodulation, i n h i b i t i o n o f DNA m e t h y l a t i o n , etc. The e v a l u a t i o n o f p o t e n t i a l c a r c i n o g e n i c i t y b a s e d on e p i g e n e t i c mechanism, r e q u i r e s e x a m i n a t i o n o f the following questions: ( i ) what i s the most likely mechanism(s)?, ( i i ) i s i n f o r m a t i o n a v a i l a b l e t o allow SAR a n a l y s i s b a s e d on t h i s mechanism?, and ( i i i ) are p o s s i b l y o t h e r mechanism(s) i n v o l v e d ? F o r example, a variety of peroxisome proliferators with different chemical structures have been shown to be hepatocarcinogenic (19). At l e a s t f o r the c h l o r i n a t e d p h e n o x y a c e t i c a c i d s ( 2 0 ) , some SAR c a n be d i s c e r n e d ( F i g . 2). I t appears t h a t the degree of branching a t the CO-1 carbon plus the electron-withdrawing c a p a b i l i t y of the c h l o r i n a t e d phenoxy m o i e t y c o n t r i b u t e t o t h e p e r o x i s o m e proliferative activity. Among the chlorinated p h e n o x y a c e t i c a c i d d e r i v a t i v e s t h a t have been t e s t e d f o r c a r c i n o g e n i c a c t i v i t y , two o f t h e more p o t e n t p e r o x i s o m e proliferators (ciprofibrate and clofibric acid) are carcinogenic whereas for 2,4-D (a weak peroxisome p r o l i f e r a t o r ) t h e r e a p p e a r s t o be no c o n v i n c i n g e v i d e n c e f o r c a r c i n o g e n i c i t y (21; s e e a l s o r e f s . 3,4 and this m o n o g r a p h ) . I t r e m a i n s t o be s t u d i e d w h e t h e r t h i s SAR o f p e r o x i s o m e p r o l i f e r a t i v e a c t i v i t y c a n be u s e d t o p r e d i c t

11. WOO&ARCOS

Evaluation of Pesticides for Potential Carcinogenicity

Relative Peroxisome Proliferative Activity of Chlorinated Phenoxyacetic A c i d s a n d Related C o m p o u n d s

Compound

R.P.*

CH o : cooH


3

c,

"i>-

Role of StructureActivity Relationship Analysis in Evaluation of

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