Probing Bioactive Mechanisms - ACS Publications - American

powerful tool in the design of new drugs (7). Symptomatology, .... a variety of aqueous organic solvent systems and found that for many ... 1987 E l s...
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Chapter 23

Base-Line Toxicity Predicted by Quantitative Structure—Activity Relationships as a Probe for Molecular Mechanism of Toxicity Downloaded via TUFTS UNIV on July 7, 2018 at 09:19:25 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Robert L. Lipnick Office of Toxic Substances (TS-796), U.S. Environmental Protection Agency, 401 M Street, SW, Washington, DC 20460 Narcosis represents the most fundamental mechanism of the toxicity of nonelectrolyte organic compounds, and corresponds to minimum or baseline toxicity. Quantitative structure-activity relationships for chemicals acting by this mechanism for various organisms and routes of exposure provide a valuable probe for determining whether or not a candidate chemical acts via narcosis or by an electrophile, proelectrophile, cyanogenic, or other more specific molecular mechanism. Structure-activity relationships (SAR) and quantitative structureactivity relationships (QSAR) have attracted increased interest by the U.S. Environmental Protection Agency (1-3) in the review of new industrial chemicals prior to their manufacture under section 5 of the Toxic Substances Control Act (TSCA) ( 4 ) . There is also a strong impetus in Europe to develop QSAR models to assess the potential toxicity of industrial organic chemicals ( 5 ) . The applicability of such predictive models is dependent upon the availability of measured or predicted values of the parameters used in the QSAR model, and by the validity of the assumption that the mechanism of toxicity of the candidate chemical is the same as that of the compounds used to derive the QSAR, and that they encompass the same range in spanned substituent space ( 6 ) . The relationships that should ideally be satisfied for such models are illustrated in Figure 1. In the absence of more specific effects, nonelectrolyte organic compounds act by a narcosis mechanism, which represents minimum or baseline toxicity. Baseline toxicity QSAR models can be used as a probe to identify chemicals acting by more specific toxicological mechanisms. Demonstrating Commonality of Molecular Mechanism The assumption of the commonality of mechanism is rarely stated in a QSAR study, but can be based implicitly or explicitly on one or more of the following: This chapter not subject to U.S. copyright Published 1989 American Chemical Society

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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367

PHYSIOLOGICAL RESPONSE

MECHANISM BASED MOLECULAR DESCRIPTORS

PREDICTION OF TOXICITY OF UNTESTED CHEMICALS

F i g u r e 1. Mechanism-based QSAR model and i t s r e l a t i o n s h i p t o c h e m i c a l and b i o l o g i c a l d a t a o f t h e m o l e c u l e s u s e d i n i t s d e r i v a t i o n . A q u a n t i t a t i v e p r e d i c t i o n o f t h e t o x i c i t y o f an u n t e s t e d c h e m i c a l r e q u i r e s t h e a v a i l a b i l i t y o f a QSAR model f o r r e l a t e d compounds a c t i n g b y a putative common m o l e c u l a r mechanism and t h e a b i l i t y t o measure o r p r e d i c t t h e r e q u i r e d mechanism-based m o l e c u l a r d e s c r i p t o r s u s e d i n t h e QSAR model.

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P R O B I N G BIOACTTVE M E C H A N I S M S

1. 2. 3. 4. 5.

Receptor b i n d i n g Symptomatology QSAR c o r r e l a t i o n Additivity of biological Chemical p r o p e r t i e s

activity

JReceptor Binding. The f i r s t c r i t e r i o n c o n s i s t s o f s i m i l a r t y p e s o f b i n d i n g w i t h i s o l a t e d r e c e p t o r s o r o t h e r m o l e c u l a r s i t e s o f a c t i o n and u n d e r s t a n d i n g o f t h e pathway o f c a s c a d i n g b i o c h e m i c a l and p h y s i o l o g i c a l p e r t u r b a t i o n s l e a d i n g t o t h e o b s e r v e d end e f f e c t . I n f o r m a t i o n from t h e e x a c t s t r u c t u r e o f and o t h e r r e c e p t o r s enzymes i s becoming an e x t r e m e l y p o w e r f u l t o o l i n t h e d e s i g n o f new drugs ( 7 ) . Symptomatology, A second criterion consists o f commonality o f syndromes, and b i o c h e m i c a l and p h y s i o l o g i c a l changes a s s o c i a t e d w i t h t h e t o x i c end e f f e c t . S i m i l a r symptomatology has p r o v i d e d a t r a d i t i o n a l c r i t e r i o n f o r a s s e s s i n g t h e d e g r e e t o w h i c h a group o f compounds may be a c t i n g by a common mechanism, and as e v i d e n c e f o r a change i n mechanism within a series. Chemicals producing a narcosis or anesthetic p h y s i o l o g i c a l r e s p o n s e a r e f r e q u e n t l y c l a s s i f i e d o n t h i s b a s i s due t o t h e l a c k o f a s p e c i f i c r e c e p t o r t h a t has been c h a r a c t e r i z e d a t t h e molecular level. More recently an attempt h a s been made t o m a t h e m a t i c a l l y t r a n s f o r m a l a r g e number o f q u a n t i t a t i v e symptomatology p a r a m e t e r s t o a s m a l l e r number o f o r t h o g o n a l d e s c r i p t o r s i n w h i c h symptomatology syndromes c o u l d be d e f i n e d t h r o u g h c l u s t e r a n a l y s i s ( f i ­ ll). QSAR Correlation. S t a t i s t i c a l q u a l i t y o f QSAR c o r r e l a t i o n c a n be employed as a t h i r d c r i t e r i o n o f commonality o f mechanism. This a p p r o a c h c a n p r o v e v e r y m e a n i n g f u l when c o u p l e d w i t h a m e c h a n i s t i c i n t e r p r e t a t i o n o f t h e r o l e o f molecular d e s c r i p t o r s used i n the c o r r e l a t i o n , and w i t h t h e s i g n i f i c a n c e o f t h e s l o p e and i n t e r c e p t . The q u a l i t y o f s t a t i s t i c a l f i t and t h e i n t e r p r e t a t i o n o f t h e p a r a m e t e r o r parameters used i n the c o r r e l a t i o n can p r o v i d e a v a l u a b l e i n s i g h t i n t o m o l e c u l a r mechanism. R e c e n t l y , Hansch a n a l y s i s has been combined w i t h m o l e c u l a r g r a p h i c s and m o d e l i n g s t u d i e s i n w h i c h t h e a c t i v i t i e s o f a s e r i e s o f s u b s t r a t e s t o an enzyme r e c e p t o r have been r e l a t e d t o t h e h y d r o p h o b i c , e l e c t r o n i c , and s t e r i c r e q u i r e m e n t s f o r r e v e r s i b l e b i n d i n g (12). Additivity of Biological Activity. Additivity of t o x i c i t y i s a valuable means f o r d e m o n s t r a t i n g commonality o f mechanism. I n most c a s e s , compounds a c t i n g b y d i f f e r e n t mechanisms show t o x i c i - t i e s t h a t a r e l e s s t h a n a d d i t i v e ( 1 3 ) . I t has been d e m o n s t r a t e d t h a t t h e a q u a t i c t o x i c i t y o f up t o 50 n o n e l e c t r o l y t e o r g a n i c c h e m i c a l s a c t i n g by a n a r c o s i s mechanism e x h i b i t s s t r i c t l y a d d i t i v e b e h a v i o r (14-16). Chemical Properties. F i n a l l y , s i m i l a r i t y i n c h e m i c a l p r o p e r t i e s and r e a c t i v i t y b a s e d upon m e c h a n i s t i c and p h y s i c a l o r g a n i c c h e m i s t r y c a n a l s o be u s e d t o s u p p o r t a h y p o t h e s i s o f commonality o f mechanism ( 1 7 ) . By t h i s means, a t r a i n e d o r g a n i c c h e m i s t c a n s u g g e s t t h e l i m i t a t i o n s o f a mechanism even f o r d i s t a n t l y r e l a t e d compounds b a s e d upon t h e i r

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UPNICK

369

c h e m i c a l p r o p e r t i e s , thus p e r m i t t i n g t h e i d e n t i f i c a t i o n t h a t c a n p o t e n t i a l l y a c t b y more s p e c i f i c mechanisms. Discovery Solubility

of the Correlation and Molecular Weight

Between

Narcosis

o f chemicals

Potency

and

Mater

I n t e r e s t has e x i s t e d f o r some time r e g a r d i n g what p r o p e r t y o f a m o l e c u l e i s r e s p o n s i b l e f o r t h e p r o d u c t i o n o f a s p e c i f i c type o f b i o l o g i c a l response (28-20). I n the case o f n a r c o s i s o r a n e s t h e t i c response, t h i s was c o m p l i c a t e d b y t h e f i n d i n g t h a t n a r c o s i s c o u l d be p r o d u c e d b y a wide v a r i e t y o f d i f f e r e n t , a p p a r e n t l y u n r e l a t e d compounds, i n c l u d i n g s i m p l e saturated monohydric alcohols. Perhaps the e a r l i e s t systematic i n v e s t i g a t i o n o f t h e m e c h a n i s t i c b a s i s o f n a r c o s i s was t h a t o f C r o s a t t h e U n i v e r s i t y o f S t r a s b o u r g , who i n 1863 r e p o r t e d t h a t t o x i c i t y o f s i m p l e a l c o h o l s a d m i n i s t e r e d t o mammals i n c r e a s e s w i t h d e c r e a s i n g w a t e r s o l u b i l i t y , up t o a p o i n t o f maximum p o t e n c y , beyond w h i c h i t d e c r e a s e s , u n t i l t h e a l c o h o l s become v e r y i n s o l u b l e and a c t l i k e f a t t y s u b s t a n c e s (21). R i c h a r d s o n i n E n g l a n d (22) i n 1869 found t h a t t h e t o x i c i t y o f simple monohydric a l c o h o l s t o mammals and b i r d s increased with i n c r e a s i n g m o l e c u l a r w e i g h t , and t h i s r e l a t i o n s h i p h a s been r e f e r r e d t o as R i c h a r d s o n ' s l a w ( 2 3 ) . Rabuteau i n F r a n c e (24) i n 1870 c o n c l u d e d t h a t t h e i n c r e a s e i n t h e t o x i c i t y t o f r o g s immersed i n s o l u t i o n s o f a l c o h o l s r e f l e c t e d an i n c r e a s e i n c h a i n l e n g t h . Both o f these s c i e n t i s t s were a p p a r e n t l y unaware o f C r o s * e a r l i e r , more f u n d a m e n t a l d i s c o v e r y . A l t h o u g h C r o s * d i s c o v e r y was c o n f i r m e d s e v e r a l y e a r s l a t e r b y D u j a r d i n Beaumetz and A u d i g e (25-27), i t was a p p a r e n t l y n o t u n t i l 1893 t h a t t h e r e l a t i o n s h i p between water s o l u b i l i t y and n a r c o s i s - p r o d u c e d t o x i c i t y was i n v e s t i g a t e d more c a r e f u l l y . Georges H o u d a i l l e , a d o c t o r a l s t u d e n t o f Charles Richet a t the U n i v e r s i t y o f P a r i s , observed a c o r r e l a t i o n between water s o l u b i l i t y and minimum n a r c o s i s c o n c e n t r a t i o n t o f i s h and t a d p o l e s f o r a v a r i e t y o f h y p n o t i c agents (28). This relationship i s f r e q u e n t l y r e f e r r e d t o as R i c h e t * s l a w ( 2 9 ) . Discovery Coefficient

of

the Correlation

of

Narcosis

Potency

and

Partition

The d i s c o v e r y o f a parameter ( o l i v e o i l / w a t e r p a r t i t i o n c o e f f i c i e n t ) upon w h i c h a m e c h a n i s t i c i n t e r p r e t a t i o n f o r n a r c o s i s c o u l d be b a s e d was made i n d e p e n d e n t l y s i x y e a r s l a t e r b y C h a r l e s E r n e s t O v e r t o n a t t h e U n i v e r s i t y o f Z u r i c h (30-31) and b y Hans H o r s t Meyer (32) and h i s c o l l a b o r a t o r F r i t z Baum (33) a t t h e U n i v e r s i t y o f Marburg. Prior to t h i s d i s c o v e r y , W a l t e r D u n z e l t , a s t u d e n t o f Meyer, attempted t o c o n f i r m t h e H o u d a i l l e d a t a o n t h e r e l a t i o n s h i p between water s o l u b i l i t y and minimum t o x i c c o n c e n t r a t i o n , u s i n g t a d p o l e s and s m a l l f i s h ( 3 4 ) . D u n z e l t c o n c l u d e d i n h i s 1896 I n a u g u r a l D i s s e r t a t i o n (34) t h a t a l t h o u g h t h i s c o r r e l a t i o n seemed t o be g e n e r a l l y c o r r e c t , i t d i d n o t h o l d f o r two compounds. Bromal h y d r a t e was f o u n d t o be v e r y s o l u b l e i n w a t e r and p r o d u c e n a r c o s i s i n f i s h a t low c o n c e n t r a t i o n s . By c o n t r a s t , m e t h y l u r e t h a n e was found t o p r o d u c e o n l y a s l i g h t n a r c o t i c e f f e c t , even though i t was o n l y s l i g h t l y s o l u b l e i n w a t e r . T h i s f i n d i n g o f D u n z e l t may have p r o v i d e d Meyer w i t h t h e impetus t o s e a r c h f o r a n o t h e r c h e m i c a l property t h a t b e t t e r c o r r e l a t e d with n a r c o t i c potency. Meyer f u r t h e r

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supported the r e l e v a n c e o f p a r t i t i o n c o e f f i c i e n t t o the m e c h a n i s t i c b a s i s o f n a r c o s i s by d e m o n s t r a t i n g t h a t t h e t e m p e r a t u r e dependence o f n a r c o s i s i n t a d p o l e s f o l l o w e d t h e same t r e n d s as t h e c o r r e s p o n d i n g o l i v e o i l / w a t e r p a r t i t i o n c o e f f i c i e n t s (35). O v e r t o n ' s d i s c o v e r y e v o l v e d from s t u d i e s o f c e l l p e r m e a b i l i t y w i t h a l g a e , and l a t e r , t a d p o l e s (36-37). Overton p r o v i d e d c o n s i d e r a b l e s u p p o r t two y e a r s l a t e r f o r t h i s l i p o i d t h e o r y o f n a r c o s i s i n h i s c l a s s i c monograph " S t u d i e n iiber d i e N a r k o s e , " (32) whose f i n d i n g s have r e c e n t l y been r e v i e w e d (38). O v e r t o n ' s t a d p o l e d a t a have been employed i n s e v e r a l QSAR s t u d i e s (39-42) ( L i p n i c k , R.L. I n QSAR in Drug Design; F a u c h e r e , J . L . , Ed.; A l a n R. L i s s : New Y o r k , i n p r e s s . ) . More Recent

Studies

of

Narcosis

For the next f i v e decades, the c o r r e l a t i o n o f p a r t i t i o n c o e f f i c i e n t w i t h n a r c o s i s p o t e n c y c o n t i n u e d t o be an i m p o r t a n t a r e a f o r r e s e a r c h , w i t h s t u d i e s o f v a r i o u s compounds p e r f o r m e d u s i n g whole o r g a n i s m s , organs, c e l l s , and enzymes. T h i s work has been d i s c u s s e d i n a number o f major r e v i e w s ( 4 2 - 5 2 ) , and i n p a p e r s from an i n t e r n a t i o n a l c o n f e r e n c e on t h i s s u b j e c t h e l d i n P a r i s i n 1950 ( 5 2 ) . D e s p i t e t h e c o n s i d e r a b l e number o f s t u d i e s o f t h e mechanism o f n a r c o s i s , t h e d e t a i l s r e g a r d i n g i t s m o l e c u l a r b a s i s have p r o v e n v e r y e l u s i v e , and t h e t h e o r e t i c a l b a s i s f o r n a r c o s i s o r a n e s t h e s i a s t i l l r e p r e s e n t s a s u b j e c t o f c o n s i d e r a b l e debate. Current research i s f o c u s e d on two major t h e o r i e s , d i s o r g a n i z a t i o n o f membrane l i p o i d c o n s t i t u e n t s (53-54), and b i n d i n g t o one o r more enzymes o r p r o t e i n s (55-56). Linear

QSAR Models

for

Narcosis

Baseline

Toxicity

In t h e mid 1960s Hansch and co-workers employed r e g r e s s i o n a n a l y s i s t o develop quantitative structure-activity relationships (57). They r e p o r t e d a number o f l i n e a r QSARs f o r s i m p l e n o n e l e c t r o l y t e s a c t i n g by a n a r c o s i s mechanism ( 4 0 ) , i n t h e form, log

(1/C) - A l o g P + B

(1)

where C i s t h e m o l a r c o n c e n t r a t i o n p r o d u c i n g a s t a n d a r d b i o l o g i c a l r e s p o n s e , and P i s t h e n - o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t . When an o r g a n i s m i s p l a c e d i n an aqueous s o l u t i o n o f a t o x i c a n t , p s e u d o - s t e a d y - s t a t e p a r t i t i o n i n g t a k e s p l a c e between t h e t o x i c s i t e o f a c t i o n and t h e aqueous p h a s e . F o r organisms i n w h i c h a i r i s e x t r a c t e d from t h e w a t e r by means o f g i l l s , exchange o f t o x i c a n t t a k e s p l a c e m a i n l y v i a t h i s r o u t e , and t h e r a t e o f u p t a k e i s c o n t r o l l e d by t h e c r o s s - s e c t i o n a l a r e a , and t h e r a t e o f b l o o d f l o w a c r o s s t h e g i l l s . R e c e n t l y , a number o f i n v e s t i g a t o r s have r e p o r t e d l i n e a r QSARs f o r the t o x i c i t y o f simple n o n e l e c t r o l y t e s t o a q u a t i c organisms (58-63). A g e n e r a l r e p r e s e n t a t i o n o f s u c h QSARs and t h e i r l i m i t a t i o n s i s d e p i c t e d i n F i g u r e 2. These e q u a t i o n s a r e o f enormous v a l u e t o EPA and o t h e r r e g u l a t o r y agencies i n s e t t i n g t e s t i n g p r i o r i t i e s f o r chemicals i n commerce and f o r new i n d u s t r i a l c h e m i c a l s p r i o r t o t h e i r m a n u f a c t u r e . A c o r r e s p o n d i n g p s e u d o - s t e a d y - s t a t e p a r t i t i o n i n g t a k e s p l a c e between t h e t o x i c a n t v a p o r and t h i s same s i t e o f a c t i o n f o r gaseous a n e s t h e t i c

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DOMAIN OF EXCESS TOXICITY

BASELINE NARCOSIS MECHANISM QSAR

THEORETICAL HoO SOLUBILITY CUTOFF. LIQUID SOLUTES LOCAL QSAR ELECTROPHILE AND PROELECTROPHILE MECHANISMS

PHARMACOKINETIC H5O SOLUBILITY CUTOFF OF BILINEAR OSARs H2O SOLUBILITY LIQUID SOLUTES

Log P F i g u r e 2. Baseline narcosis aquatic t o x i c i t y QSAR model. For n o n e l e c t r o l y t e s a c t i n g b y a n a r c o s i s mechanism, no t o x i c i t y i s o b s e r v e d i f t h e p r e d i c t e d t o x i c c o n c e n t r a t i o n exceeds the w a t e r s o l u b i l i t y . W i t h decreasing t e s t d u r a t i o n , pseudo-s t e a d y - s t a t e p a r t i t i o n i n g i s not a c h i e v e d f o r v e r y h y d r o p h o b i c c h e m i c a l s , and t h e l o c a t i o n o f t h e water solubility cutoff shifts t o chemicals having a lower partition coefficient. Compounds a c t i n g b y more s p e c i f i c mechanisms p r o d u c e t o x i c i t y a t l o w e r aqueous c o n c e n t r a t i o n s t h a n p r e d i c t e d b y n a r c o s i s , and f a l l w i t h i n t h e domain o f e x c e s s t o x i c i t y .

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agents. I n t h i s c a s e , t h e o i l / g a s p a r t i t i o n c o e f f i c i e n t and n o t t h e o i l / w a t e r p a r t i t i o n c o e f f i c i e n t i s t h e a p p r o p r i a t e model p a r a m e t e r and y i e l d s a l i n e a r c o r r e l a t i o n as shown i n F i g u r e 3. By c o n t r a s t , o n l y s c a t t e r i s o b s e r v e d i f o c t a n o l / w a t e r i s u s e d as t h e model p a r a m e t e r as shown i n F i g u r e 4 ( 6 4 ) . Model Partitioning

Systems

O v e r t o n and Meyer b o t h u s e d o l i v e o i l as a p a r t i t i o n i n g system t o model t h e p h y s i c o c h e m i c a l p r o p e r t i e s o f t h e p u t a t i v e membrane l i p o i d s i t e o f a c t i o n . A l t h o u g h O v e r t o n attempted t o u s e m e l t e d c h o l e s t e r o l and o t h e r s u b s t a n c e s he t h o u g h t might s e r v e as a b e t t e r r e f e r e n c e phase, he abandoned this a p p r o a c h due t o problems w i t h the formation of i n s e p a r a b l e e m u l s i o n s ( 3 1 ) . C o l l a n d e r i n F i n l a n d (65) e x p e r i m e n t e d w i t h a v a r i e t y o f aqueous o r g a n i c s o l v e n t systems and f o u n d t h a t f o r many s i m p l e n o n e l e c t r o l y t e s , t h e v a l u e s were w e l l - c o r r e l a t e d a c c o r d i n g t o t h e following equation; l o g Pi « a l o g P

2

+ b

(2)

where Pi and ?£ a r e two s u c h p a r t i t i o n i n g s y s t e m s . The C o l l a n d e r e q u a t i o n s were i n v e s t i g a t e d mora f u l l y by Leo and Hansch. They c o n c l u d e d t h a t s o l u t e s a b l e t o a c t as e i t h e r s t r o n g hydrogen-bond donors o r a c c e p t o r s r e q u i r e an a d d i t i o n a l c o r r e c t i o n t o a c c o u n t f o r t h e r e l a t i v e a b i l i t y o f the o r g a n i c s o l v e n t s t o i n t e r a c t i n t h i s f a s h i o n w i t h r e s p e c t t o one a n o t h e r ( 6 6 ) . A l t h o u g h n - o c t a n o l i s now commonly u s e d as a model o r g a n i c phase, i t s h o u l d be k e p t i n mind t h a t i t may be a b e t t e r model f o r some b i o l o g i c a l systems t h a n f o r o t h e r s . Meyer and Hemmi (67) compared t h e a b i l i t y o f p a r t i t i o n c o e f f i c i e n t s from d i f f e r e n t s o l v e n t systems t o model n a r c o s i s d a t a , and t h e y f o u n d o l e y l a l c o h o l t o y i e l d the best r e s u l t s . F o r complex o r g a n i c m o l e c u l e s c o n t a i n i n g h y d r o g e n b o n d i n g f u n c t i o n a l g r o u p s , t h e c h o i c e o f a p a r t i c u l a r model s y s t e m c o u l d l e a d t o a p p a r e n t o u t l i e r s w h i c h may n o t r e f l e c t a t r u e change o f mechanism a t t h e m o l e c u l a r s i t e o f a c t i o n . Estimation

of Partition

Coefficients

The development o f t h e fragment c o n s t a n t methodology f o r p r e d i c t i n g log P values directly from chemical s t r u c t u r e (68-71) and i t s c o m p u t e r i z a t i o n (72-73) i s f u r t h e r i n g t h e use o f n - o c t a n o l as a s t a n d a r d r e f e r e n c e p h a s e . A n o t h e r a p p r o a c h t h a t has been p u r s u e d i s t h e use o f r e t e n t i o n t i m e s d e t e r m i n e d on a r e v e r s e d - p h a s e HPLC column t o e s t i m a t e l o g P, u s i n g a s e t o f s t a n d a r d s o l u t e s f o r r e f e r e n c e ( 7 4 - 7 6 ) . These c o r r e l a t i o n s d i s p l a y l i m i t a t i o n s s i m i l a r t o t h o s e i n c h a n g i n g from one p a r t i t i o n i n g system t o another i f hydrogen-bonding i n t e r a c t i o n s are significant. The i n t e r a c t i o n s o f t h i s t y p e o f s o l u t e w i t h b i o l o g i c a l s u b s t r a t e s have a l s o been modeled u s i n g s o l v a t o c h r o m i c p a r a m e t e r s ( 7 7 ) . Species

Sensitivity

to

Narcosis

G e n e r a l l y , t h e s l o p e s o f QSARs f o r n a r c o s i s a r e c l o s e t o u n i t y i n d i c a t i n g t h a t (1) n - o c t a n o l p r o v i d e s a r e a s o n a b l e model f o r t h e p h y s i c o c h e m i c a l p r o p e r t i e s o f t h e b i o l o g i c a l s i t e o f a c t i o n , (2) p s e u d o -

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

23. LIPNICK

Base-Line Toxicity Predicted by QSAR

0

0.5

1

1.5

2

373

2.5

Log K (Oil/Gas) F i g u r e 3. L i n e a r r e l a t i o n s h i p between o i l / g a s p a r t i t i o n c o e f f i c i e n t and minimum c o n c e n t r a t i o n i n a i r r e q u i r e d t o produce a n e s t h e s i a i n mice on a log log scale. (Reproduced w i t h p e r m i s s i o n from R e f . 64. C o p y r i g h t 1987 E l s e v i e r ) .

F i g u r e 4. L a c k o f a r e l a t i o n s h i p between o c t a n o l / w a t e r partition c o e f f i c i e n t and minimum c o n c e n t r a t i o n i n a i r r e q u i r e d t o p r o d u c e a n e s t h e s i a i n mice on a l o g l o g s c a l e . (Reproduced w i t h p e r m i s s i o n from R e f . 64. C o p y r i g h t 1987 E l s e v i e r ) .

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

374

PROBING BIOACTIVE

MECHANISMS

s t e a d y - s t a t e p a r t i t i o n i n g has been a c h i e v e d , and (3) any l o s s o f t o x i c a n t v i a metabolism i s reasonably constant w i t h i n the s e r i e s s t u d i e d . The i n t e r c e p t i n QSAR i s a measure o f t h e r e l a t i v e s e n s i t i v i t y of the organism. B o t h Meyer and O v e r t o n p o s t u l a t e d t h a t t h e r e l a t i v e s e n s i t i v i t y o f chemicals i s r e l a t e d t o the percent o f l i p o i d t i s s u e w i t h i n t h e o r g a n i s m , h i g h e r organisms b e i n g t h e most s e n s i t i v e due t o the h i g h c o n c e n t r a t i o n o f s u c h l i p o i d s i n n e r v e c e l l s . I n comparing r e l a t i v e s e n s i t i v i t i e s , i t i s e s s e n t i a l t h a t a common p h y s i o l o g i c a l e n d p o i n t be employed. Thus, QSAR e q u a t i o n s d e v e l o p e d from d a t a o n m u l t i - g e n e r a t i o n a l t o x i c i t y t o a l g a e , p r o t o z o a , and b a c t e r i a have i n t e r ­ cepts t h a t r e f l e c t higher s e n s i t i v i t y than simple n a r c o s i s (78). D a t a from Fuhner (79) o n t h e n a r c o s i s by a l c o h o l s t o 23 d i f f e r e n t o r g a n i s m s r e p r e s e n t i n g a b r o a d range i n taxonomic c l a s s were a n a l y z e d t o examine t h e M e y e r - O v e r t o n h y p o t h e s i s . Two o f t h e o r g a n i s m s i n t h i s d a t a s e t were t e s t e d w i t h a s u f f i c i e n t number o f compounds t o s a t i s f y the T o p l i s s c r i t e r i o n (80) f o r s t a t i s t i c a l v a l i d i t y w i t h r e s p e c t t o t h e number o f t e s t s p e r p a r a m e t e r . The r e m a i n i n g o r g a n i s m s were t e s t e d u s i n g o n l y e t h a n o l and n - h e p t a n o l . N e v e r t h e l e s s , t h e u s e o f o n l y two p o i n t s p e r o r g a n i s m c a n be j u s t i f i e d i n t h i s c a s e b y t h e o b s e r v a t i o n o f common symptomatology, a s p e c i f i c p h y s i c o c h e m i c a l i n t e r p r e t a t i o n o f t h e l o g F p a r a m e t e r , and g e n e r a l a s s o c i a t i o n o f s a t u r a t e d monohydric a l c o h o l s w i t h a n a r c o s i s mechanism. The s l o p e s and i n t e r c e p t s d e r i v e d from t h e s e data o f Fuhner a r e a r r a n g e d i n order of increasing s e n s i t i v i t y i n Table I . T h e r e i s an e v i d e n t association of greater s e n s i t i v i t y with i n c r e a s i n g p h y l o g e n e t i c development. The p a r t i a l o v e r l a p between i n v e r t e b r a t e s and v e r t e b r a t e s may r e f l e c t d i f f e r e n c e s i n t h e s p e c i f i c b i o l o g i c a l e n d p o i n t c h o s e n b y Fuhner f o r o b s e r v a t i o n . The i n t e r c e p t s o b t a i n e d f o r f i s h and t a d p o l e s a r e s i m i l a r t o t h o s e r e p o r t e d i n t h e l i t e r a t u r e b a s e d upon o t h e r t e s t d a t a ( 8 2 - 8 2 ) .

Water solubility

cutoff

Pseudo-steady-state p a r t i t i o n i n g can take p l a c e i n a q u a t i c organisms immersed i n an aqueous s o l u t i o n o f t o x i c a n t o r v i a e x p o s u r e t o v a p o r by i n h a l a t i o n through the lungs. The c o n c e n t r a t i o n o f a t o x i c a n t a t t h e n a r c o s i s s i t e o f a c t i o n i s a f u n c t i o n o f b o t h i t s aqueous c o n c e n t r a t i o n and p a r t i t i o n c o e f f i c i e n t . I n c r e a s i n g l y l o w e r aqueous c o n c e n t r a t i o n s a r e r e q u i r e d t o p r o d u c e a common m o l a r c o n c e n t r a t i o n a t t h e s i t e o f action with increasing p a r t i t i o n c o e f f i c i e n t . These r e l a t i o n s h i p s a r e i l l u s t r a t e d i n F i g u r e 2. O v e r t o n p o i n t e d o u t i n h i s c l a s s i c monograph " S t u d i e n iiber d i e N a r k o s e " t h a t a c l a s s i f i c a t i o n e x i s t e d a t t h a t time o f s u b s t a n c e s w h i c h n a r c o t i z e b o t h p l a n t s and a n i m a l s ( 3 2 , 3 8 ) , and t h o s e s u c h as s u l f o n a l that n a r c o t i z e only animals. O v e r t o n p r o v i d e d a much s i m p l e r and more reasonable e x p l a n a t i o n f o r t h i s apparent dichotomy. The n a r c o t i z i n g c o n c e n t r a t i o n o f s u l f o n a l t o tadpoles i s v e r y c l o s e t o t h e water s o l u b i l i t y o f t h i s compound. O v e r t o n f o u n d t h a t a l g a e r e q u i r e d 6-7 times as g r e a t a c o n c e n t r a t i o n t o p r o d u c e n a r c o s i s as t a d p o l e s and o t h e r h i g h e r a q u a t i c o r g a n i s m s , and t h e r e f o r e r e q u i r e c o n c e n t r a t i o n s o f s u l f o n a l e x c e e d i n g i t s water s o l u b i l i t y t o p r o d u c e n a r c o s i s .

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Base-Line Toxicity Predicted by QSAR

LIPNICK

Table

375

I . R e l a t i o n s h i p Between S p e c i e s S e n s i t i v i t y t o N a r c o s i s and Taxonomic H i e r a r c h y 3

Species

Type

Noctiluca miliaris

Protozoa

0.33

Covoluta roseoffensis

Sea u r c h i n

0.63 0.64^

Physa fontinalis

Freshwater snail

0.65

1.04

Locomotion

Tomopteris onisciformis

Worm

0.70

1.00

Suppress swimming

Pec ten operculus

Small mussel

0.72

1.01

Muscle contraction on c o n t a c t

Soles (cutlellus) pellucidus

Mussel

0.72

1.01

Muscle contraction on c o n t a c t

Asterias rubens

Sea

0.72

1.01

Locomotion

Worm

0.72

1.01

Locomotion

Gammarus pulex

Arthropod

0.73

1.03

Swimming

My sis (macromysis) flexuosa

Marine crab

0.73

1.03

Swimming

Aeolis drummondi and rufibranchialis

Marine n i g h t snail

0.74

1.06

Locomotion

Amphioxus vulgaris

Acranian

0.75

1.13

Swimming o n contact

Spio

vulgaris

Sensitivity (Intercept)

star

c

Slope

Narcosis Endpoint

0.96

Tentacle movement c

0.98 0.98^

Suppress movement

Continued on next page

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

376

PROBING BIOACTIVE

Table

I . Continued

Slope

Narcosis Endpoint

1.00

Swimming

0.77

1.20

Swimming

Water f r o g

0.78

1.14

Narcosis

Cyclopterus lumpus

Fish

0.80

1.20

Narcosis

Cydippe pileus

C1eonphones

0.82

0.97

Tentacle paralysis

Tadpole

0.82

1.16

Narcosis

Triton vulgaris

Water salamander

0.83

1.12

Narcosis

Rana

Tadpole

0.85

1.14

Narcosis

Sepiola rondeletti

Cephalopod

0.88

1.25

Narcosis

Actinia equina

Sea

0.90

1.02

Tentacle paralysis

Phoxinus laevis

Minnow fish

Species

Type

Idothea tricuspidata

Arthropod

Pleuronectes platessa

Fish

Rana esculenta

Rana

a

MECHANISMS

fusea

agilis

Sensitivity (Intercept)

anemone

0.75

1.08

c

0.89

c

Narcosis

b

D a t a from Fuhner ( 7 9 ) . D a t a f o r e t h a n o l ( l o g F • -0.235) and h e p t a n o l ( l o g P = 2.410) were u s e d t o a l g e b r a i c a l l y o b t a i n t h e s l o p e (A) and t h e i n t e r c e p t (B) f o r t h e e q u a t i o n l o g (1/C) = A l o g P + B, where C i s the l i m i t i n g n a r c o s i s producing concentration i n moles/L. Calculated by r e g r e s s i o n analysis from data on 5 a l c o h o l s . C a l c u l a t e d f r o m d a t a f o r e t h a n o l and h e p t a n o l . C a l c u l a t e d by r e g r e s s i o n a n a l y s i s from d a t a f o r 10 a l c o h o l s . e

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

23.

LIPNICK

Base-Line Toxicity Predicted by QSAR

377

L i m i t i n g water s o l u b i l i t y a l s o accounts f o r t h e appearance o f a c u t o f f o r a b r u p t change from t o x i c t o n o n t o x i c w i t h i n a s e r i e s o f compounds ( F i g u r e 2 ) . O v e r t o n d e m o n s t r a t e d , however, t h a t p r i o r t o t h e a p p e a r a n c e o f s u c h a c u t o f f , an i n c r e a s e i n p a r t i t i o n c o e f f i c i e n t , s u c c e s s i v e l y l o w e r c o n c e n t r a t i o n s o f t o x i c a n t a c h i e v e t h i s same e f f e c t . T h i s i s accompanied b y t h e need f o r i n c r e a s i n g t e s t d u r a t i o n t o a c h i e v e p s e u d o - s t e a d y - s t a t e p a r t i t i o n i n g between t h e s i t e o f a c t i o n and t h e aqueous t e s t s o l u t i o n . Influence

of Melting

Point

on Water Solubility

Cutoff

The w a t e r s o l u b i l i t y c u t o f f f o r an o r g a n i c compound i s a f u n c t i o n o f t h e minimum c o n c e n t r a t i o n i n d u c i n g n a r c o s i s and t h e water s o l u b i l i t y ( F i g u r e 2). The l a t t e r i s r e l a t e d t o b o t h t h e p a r t i t i o n c o e f f i c i e n t and t h e m e l t i n g p o i n t o f t h e compound ( 8 3 ) . M e l t i n g p o i n t i s a measure o f t h e e n t h a l p y o f f u s i o n and r e f l e c t s t h e s t r e n g t h o f i n t e r m o l e c u l a r f o r c e s present i n the c r y s t a l l i n e state. These f o r c e s a r e c o n s i d e r a b l y s t r e n g t h e n e d by t h e p r e s e n c e o f h y d r o g e n bonds. Moreover, compounds containing a l i p h a t i c chains and o t h e r f u n c t i o n a l groups o f f e r i n g m u l t i p l e d e g r e e s o f c o n f o r m a t i o n a l freedom and l a c k i n g symmetry must l o s e t h e s e d e g r e e s o f freedom i n p a s s i n g f r o m t h e l i q u i d t o t h e s o l i d s t a t e (84-85). F o r example, phenanthrene and a n t h r a c e n e a r e i s o m e r s h a v i n g t h e same p a r t i t i o n c o e f f i c i e n t ( l o g P = 4.49). W h i l e t h e f o r m e r p r o d u c e d n a r c o s i s a t 0.0112 mmoles/L, t h e l a t t e r showed no e f f e c t a t saturation, regardless o f the duration o f exposure (37,40). Phenanthrene p o s s e s s e s a l o w e r degree o f symmetry t h a n a n t h r a c e n e , and a c o n s i d e r a b l y lower m e l t i n g p o i n t . The f o r m e r m e l t s a t 98°, and t h e l a t t e r a t 218° ( 3 7 ) . Bilinear

Relationships

and Pharmacokinetic

Cutoff

The t o x i c i t y o f c h e m i c a l s t o a q u a t i c organisms a c t i n g by a n a r c o s i s mechanism e x h i b i t s l i n e a r QSAR r e l a t i o n s h i p s w i t h t e s t s o f s u f f i c i e n t duration t o achieve pseudo-steady-state p a r t i t i o n i n g between t h e aquarium aqueous donor phase and t h e s i t e o f a c t i o n w i t h i n t h e f i s h . For s h o r t e r t e s t durations, a higher concentration i s r e q u i r e d t o permit t h e needed i n t e r n a l t o x i c c o n c e n t r a t i o n t o be a c h i e v e d , r e s u l t i n g i n a n o n - l i n e a r r e l a t i o n s h i p between t o x i c i t y and l o g P. Such d a t a have been f i t by a b i l i n e a r model ( 8 6 ) . T h i s r e l a t i o n s h i p i s i l l u s t r a t e d i n F i g u r e 2. I f the toxic concentration required at this shorter test d u r a t i o n exceeds t h e water s o l u b i l i t y , no t o x i c i t y w i l l be o b s e r v e d i n a s a t u r a t e d s o l u t i o n , even though i t w i l l be s e e n a t l o w e r c o n c e n t r a t i o n i n t e s t s o f longer duration. Thus, p e n t a c h l o r o b e n z e n e was r e p o r t e d t o be n o n t o x i c a t s a t u r a t i o n i n a 96-hour t e s t ( 6 0 ) , b u t e x h i b i t e d a 14day LC50 o f 0.18 mg/L ( 5 8 ) . B i l i n e a r r e l a t i o n s h i p s a r e a l s o observed i n r a t o r a l LD50 ( F i g u r e 5 ) , where t h e r e i s a l s o a l a c k o f e q u i l i b r i u m p a r t i t i o n i n g between t h e s i t e o f a d m i n i s t r a t i o n and t h e s i t e o f a c t i o n (64,87). Excess

Toxicity

N a r c o s i s r e p r e s e n t s t h e most f u n d a m e n t a l m o l e c u l a r mechanism o f t h e t o x i c i t y o f n o n e l e c t r o l y t e o r g a n i c compounds. QSAR c o r r e l a t i o n s d e r i v e d

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378

PROBING BIOACTIVE MECHANISMS

2.5

2

0

I

-1

1

0 1

i

a

,„„

t

2

,.

3

t

i

4

l

5

l

6

7

8

Log P Figure 5. Bilinear relationship between o c t a n o l / w a t e r partition c o e f f i c i e n t and r a t o r a l LD50 o f s a t u r a t e d monohydric a l c o h o l s and s a t u r a t e d monoketones on a l o g l o g s c a l e . (Reproduced w i t h p e r m i s s i o n from R e f . 64. C o p y r i g h t 1987 E l s e v i e r ) .

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

23.

LIPNICK

Base-Line Toxicity Predicted by QSAR

379

from data on s a t u r a t e d monohydric a l c o h o l s and o t h e r c l a s s e s wella s s o c i a t e d w i t h a n a r c o s i s mechanism c a n be u s e d as a p r o b e t o examine t h e t o x i c o l o g i c a l b e h a v i o r o f o t h e r n o n e l e c t r o l y t e o r g a n i c compounds (Figure 2 ) . Mechanistic organic chemistry provides a r a t i o n a l basis for u n c o v e r i n g t h e m o l e c u l a r mechanism o f n o n e l e c t r o l y t e s more t o x i c t h a n predicted. N o n e l e c t r o l y t e o r g a n i c compounds a c t i n g b y a more s p e c i f i c mechanism c a n be i d e n t i f i e d b y comparing t h e i r t o x i c i t y w i t h that p r e d i c t e d by a baseline n a r c o s i s equation, according to the f o l l o w i n g equation, T

e

~

C

pred / o b s

(

C

3

)

where T i s t h e excess t o x i c i t y parameter ( 6 3 ) , and C and C are t h e b a s e l i n e QSAR p r e d i c t e d a n d o b s e r v e d t o x i c i t i e s , r e s p e c t i v e l y . T h e e x c e s s t o x i c i t y o f most n o n e l e c t r o l y t e s c a n be r a t i o n a l i z e d i n terms of an e l e c t r o p h i l e or proelectrophile m o l e c u l a r mechanism, using m e c h a n i s t i c o r g a n i c c h e m i s t r y as a b a s i s f o r p r e d i c t i n g s u c h r e a c t i v i t y (82,88-89). e

p r e d

o b s

Electrophile Mechanism. E l e c t r o p h i l e t o x i c a n t s undergo d i r e c t c o v a l e n t bond f o r m a t i o n w i t h n u c l e o p h i l i c f u n c t i o n a l groups s u c h as s u l f h y d r y l t h a t a r e p r e s e n t i n enzymes a n d o t h e r c r i t i c a l b i o l o g i c a l m a c r o m o l e cules. A schematic r e p r e s e n t a t i o n o f the b i o c h e m i c a l and p h y s i o l o g i c a l processes i n v o l v e d are i l l u s t r a t e d i n Figure 6. E l e c t r o p h i l i c i t y has b e e n a s s o c i a t e d f o r some t i m e w i t h b i o l o g i c a l a c t i v i t y ( 9 2 ) . T h e c o m p o u n d s p - n i t r o b e n z y l p y r i d i n e (90) a n d p - n i t r o t h i o p h e n o l (92) h a v e been employed as model n u c l e o p h i l e s , and t h e p s e u d o - f i r s t o r d e r r e a c t i o n r a t e s have been u s e d as p a r a m e t e r s t o c o r r e l a t e w i t h e l e c t r o p h i l i c toxicity. Examples o f three types o f e l e c t r o p h i l e t o x i c a n t s , those u n d e r g o i n g d i r e c t n u c l e o p h i l i c d i s p l a c e m e n t , M i c h a e l - t y p e a d d i t i o n , and S c h i f f base f o r m a t i o n w i t h amino g r o u p s , s u c h as e-amino groups o f l y s i n e a r e shown i n T a b l e I I . Within a series of electrophiles bearing a common reactive f u n c t i o n a l group i n a s i m i l a r e l e c t r o n i c environment, excess t o x i c i t y d e c r e a s e s w i t h i n c r e a s i n g p a r t i t i o n c o e f f i c i e n t . Thus, acetaldehyde has a n e x c e s s t o x i c i t y o f 1 3 1 , a n d b u t y r a l d e h y d e , 45 ( 8 8 ) . A m o d i f i c a t i o n i n the e l e c t r o n i c environment o f a f u n c t i o n a l group t h a t r e s u l t s i n i n c r e a s e d e l e c t r o p h i l i c i t y can produce an i n c r e a s e d excess toxicity, e v e n f o r a compound w i t h a h i g h e r p a r t i t i o n c o e f f i c i e n t . Thus, pentafluorobenzaldehyde has both a h i g h e r l o g F and g r e a t e r excess toxicity than trimethoxybenzaldehyde. The f o r m e r , however, is considerably more r e a c t i v e due t o t h e p r e s e n c e of five strongly e l e c t r o n w i t h d r a w i n g groups compared t o t h r e e e l e c t r o n d o n a t i n g groups i n the l a t t e r ( 8 8 ) . There i s a s i m i l a r t r e n d i n comparing the M i c h a e l type acceptors acrolein, 2-hydroxye thy1 a c r y l a t e , and a c r y l a m i d e . L i k e w i s e , a l l y l b r o m i d e a n d a l l y l c h l o r i d e h a v e a l m o s t t h e same l o g P v a l u e , b u t d i f f e r g r e a t l y i n t h e i r e x c e s s t o x i c i t y due t o t h e g r e a t e r a b i l i t y o f bromide r e l a t i v e t o c h l o r i d e t o a c t as l e a v i n g group ( 6 3 ) . Work i s i n p r o g r e s s i n quantifying these differences based upon electronic molecular descriptors. Proelectrophile Mechanism. P r o e l e c t r o p h i l e s , which produce electro­ p h i l e s b y means o f m e t a b o l i c t r a n s f o r m a t i o n , r e p r e s e n t a s e c o n d c l a s s

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

CHEMICAL IN AQUARIUM WATER

TRANSPORT TO METABOLIZING ENZYMES

CHE M IC A L IN FISH BLOOD

by n a r c o s i s ,

COVALENT BOND FORMATION WITH CRITICAL N U M B E R OF TARGET MOLECULES

LOSS O F BIOCHEMICAL ANO PHYSIOLOGICAL FUNCTION

e l e c t r o p h i l e , and p r o e l e c t r o p h i l e

toxicants mechanisms.

t o f i s h from

o f t h e p h y s i o l o g i c a l and b i o c h e m i c a l

I

TRANSPORT TO ELECTROPHILE SITE O F ACTION

REACTION OF ELECTROPHILE WITH NUCLEOPHILIC MOIETIES O F TARGET BIOLOGICAL MACROMOLECULES

BIOCHEMICAL CHAIN O F EVENTS

i nthe production o f l e t h a l i t y

Schematic r e p r e s e n t a t i o n

processes involved

acting

W

TRANSPORT TO ELECTROPHILE , SITE OF 1 ACTION

METABOLIC ACTIVATION OF PROELECTROPHILE TO REACTIVE ELECTROPHILE MOLECULAR SPECIES

^ ~

F i g u r e 6.

GILL UPTAKE

TRANSPORT TO NARCOSIS SITE OF ACTION

Ai

CRITICAL NARCOSIS M O L A R CONCENTRATION

DEATH OF ORGANISM

00

23.

Base-Line Toxicity Predicted by QSAR

LIPNICK

381

o f n o n e l e c t r o l y t e s showing e x c e s s t o x i c i t y . Some examples o f p r o e l e c t r o p h i l e s a r e i l l u s t r a t e d i n T a b l e I I I . A l l y l a l c o h o l undergoes m e t a b o l i c o x i d a t i o n by t h e u b i q u i t o u s enzyme a l c o h o l dehydrogenase t o a c r o l e i n , a M i c h a e l - t y p e a c c e p t o r e l e c t r o p h i l e ( 6 3 ) , shown i n T a b l e I I . I t h a s been p r o p o s e d t h a t t h e e x c e s s t o x i c i t y o f 1,3-dibromopropane (88) r e f l e c t s t h e same mechanism as was p r o p o s e d f o r i t s m u t a g e n i c i t y , i . e . , m e t a b o l i s m t o t h e e l e c t r o p h i l i c f o u r membered s u l f o n i u m c a t i o n . The e x c e s s t o x i c i t y o f p e n t a e r y t h r i t o l t r i a l l y l e t h e r c a n be a c c o u n t e d f o r b a s e d upon m e t a b o l i s m v i a a f r e e r a d i c a l mechanism t o t h e c o r r e s p o n d i n g h e m i a c e t a l , which can r e a d i l y cleave t o y i e l d a c r o l e i n ( 6 3 ) . Cyanogenie Mechanism. The a c u t e t o x i c i t y o f a t h i r d c l a s s o f s i m p l e n o n e l e c t r o l y t e s c a n be r e a d i l y r a t i o n a l i z e d i n terms o f a c y a n o g e n i c mechanism, i n v o l v i n g m e t a b o l i c r e l e a s e o f c y a n i d e . Free cyanide i s h i g h l y t o x i c t o f i s h w i t h a TLm o f 0.69 mg/L (93). Some examples o f cyanogenic t o x i c a n t s a r e shown i n T a b l e IV. Lactonitrile i s f u n c t i o n a l l y s i m i l a r t o a h e m i a c e t a l , and c a n r e a d i l y l o s e c y a n i d e by a s i m p l e mechanism. I n t h e c a s e o f m a l o n o n i t r i l e and a l l y l c y a n i d e , f r e e r a d i c a l m e t a b o l i c o x i d a t i o n y i e l d s a s t a b l e s p e c i e s which on f r e e radical perhydroxylation becomes functionally equivalent to lactonitrile. Chronicity

and

Ratio.

24-Hr

LC50/96-Hr

LC50

A q u a t i c t o x i c o l o g i s t s f r e q u e n t l y r e p o r t f i s h t o x i c i t y d a t a f o r 24 96-hr d u r a t i o n s . The c h r o n i c i t y r a t i o , J?, may be d e f i n e d a s , R = LC50 _ /LC50 _ 24

h

%

h

where LC50 4_h and L C 5 0 _ a r e t h e 24-h and 96-h t o x i c i t y v a l u e s , r e s p e c t i v e l y (88). V a l u e s o f t h i s r a t i o g r e a t e r t h a n 2 have been employed b y a q u a t i c t o x i c o l o g i s t s as an i n d i c a t o r o f t h e p o t e n t i a l need to perform c h r o n i c t e s t i n g on a chemical t o assess i t s p o t e n t i a l r i s k i f r e l e a s e d i n t o t h e environment (95). An a n a l y s i s o f d a t a f o r 37 n o n e l e c t r o l y t e s d e m o n s t r a t e d a c o r r e l a t i o n between e x c e s s t o x i c i t y and a c h r o n i c i t y r a t i o g r e a t e r t h a n 2 (88). This finding i s consistent with t h e h y p o t h e s i s t h a t c h r o n i c i t y c a n r e s u l t from c o v a l e n t b i n d i n g by an e l e c t r o p h i l e o r p r o e l e c t r o p h i l e t o x i c a n t , o r by a n o t h e r more s p e c i f i c mechanism s u c h as m e t a b o l i c r e l e a s e o f c y a n i d e . These r e s u l t s a r e i l l u s t r a t e d g r a p h i c a l l y i n F i g u r e 7. A l l c h e m i c a l s i n t h i s s t u d y (88) with a T v a l u e l e s s t h a n 6.7 have a c h r o n i c i t y r a t i o (R) o f 2 o r l e s s , while the m a j o r i t y o f those with T v a l u e s g r e a t e r t h a n 6.7 show c h r o n i c i t y r a t i o s g r e a t e r t h a n 2. R r e p r e s e n t s o n l y a s i n g l e t e m p o r a l probe f o r i n v e s t i g a t i n g c u m u l a t i v e t o x i c i t y . Certain highly reactive c h e m i c a l s h a v i n g R v a l u e s l e s s t h a n 2 may p r o d u c e c u m u l a t i v e damage within shorter test durations. Conversely, i f the r a t e o f metabolic t r a n s f o r m a t i o n t o t o x i c a n t i s slow o r r e q u i r e s a complex s e r i e s o f s t e p s , l o n g e r t e s t d u r a t i o n s may be r e q u i r e d t o a s s e s s s u c h p o t e n t i a l chronicity. 2

%

h

c

e

Use of Screening Mechanism

Data

to

Investigate

the

Limitations

of

the

Narcosis

The u s e o f QSAR f o r p r e d i c t i v e purposes i s p l a c e d upon a more s e c u r e f o u n d a t i o n i f a r a t i o n a l e c a n be d e v e l o p e d f o r why t h e c a n d i d a t e

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

382

PROBING BIOACTIVE MECHANISMS

Table I I .

E l e c t r o p h i l e s and E x c e s s

Chemical

Log P Nucleophilic

-0 .792

e

490 97 >A90 35 1960 86 21 11

-0 .273 1 .590 1 .450 0 .219 3 .866 4 .440 1 .972

Michael-type

addition 0 .101 -0 .058 -0 .859

aerylate

Sehiff

T

3

Substitution

Ethylene oxide Propylene oxide A l l y l bromide A l l y l chloride Chloroacetonitrile a,a-Dichloroxylene Phenyl d i s u l f i d e 3,4-Dichloro-l-butene

Acrolein 2-Hydroxyethyl Acrylamide

Toxicity

>81000 1540 180

base f o r m a t i o n

Acetaldehyde Butyraldehyde Pentafluorobenzaldehyde a,a,a-Trifluoro-m-tolualdehyd e Benzaldehyde 2,4,5-Trime thoxybenz a1dehyde

-0 .224 0 .834 2 .449 2 .594 1 .495 1 .381

131 45 51 40 31 11

3

C a l c u l a t i o n s o f T adapted from r e f s . 63 and 88, w h i c h were based upon l i n e a r o r b i l i n e a r b a s e l i n e t o x i c i t y QSAR models. e

Table I I I .

Proelectrophiles

Chemical

and E x c e s s

Log P

A l l y l alcohol 1,3-Dibromopropane Pentaerythritol t r i a l l y l

ether

-0.250 1.987 -1.599

Toxicity

T

e

16000 87 18000

3

C a l c u l a t i o n s o f T adapted from r e f s . 63 and 88, w h i c h were based upon l i n e a r o r b i l i n e a r b a s e l i n e t o x i c i t y QSAR models. e

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3

23.

LIPNICK

Base-Line Toxicity Predicted by QSAR

383

T a b l e I V . Cyanogenic N o n e l e c t r o l y t e s and E x c e s s Toxicity

Chemical

Log P

3

Lactonitrile Malononitrile^ A l l y l cyanide

-0. 850 -1. 198 0 .120

a

c

Data from 93. ° D a t a from 94. 96-h LC50 v a l u e s .

Average

**e

23800 88700 16

c

o f three

c h e m i c a l i s l i k e l y t o a c t by t h e same mechanism as t h o s e c h e m i c a l s used to d e r i v e the e q u a t i o n . I n the case o f n o n r e a c t i v e n o n e l e c t r o l y t e s , the i d e n t i f i c a t i o n o f r e l a t e d compounds t h a t have been r e p o r t e d t o produce n a r c o s i s , a n e s t h e s i a , o r a c t as h y p n o t i c s (96) c a n be v a l u a b l e i n e s t a b l i s h i n g t h e l i k e l i h o o d o f a b a s e l i n e n a r c o s i s mechanism. Fish t o x i c i t y s c r e e n i n g d a t a have p r o v e n e s p e c i a l l y u s e f u l i n a s s e s s i n g t h e l i m i t a t i o n s o f t h e n a r c o s i s QSAR b a s e l i n e t o x i c i t y model t o t h e s e organisms ( 9 7 ) . D a t a r e t r i e v e d from f o u r s u c h s t u d i e s (98-101) were u s e d t o i n v e s t i g a t e t h e u t i l i t y and l i m i t a t i o n s o f t h e n a r c o s i s model f o r a l c o h o l s c o n t a i n i n g no o t h e r h e t e r o a t o m f u n c t i o n a l g r o u p s . A l t h o u g h t h e d a t a f o r most o f t h e s e compounds p r o v e d t o be c o n s i s t e n t w i t h t h e model, p r i m a r y and s e c o n d a r y p r o p a r g y l i c a l c o h o l s were f o u n d t o show excess t o x i c i t y . Acetylenic Alcohols. The f i n d i n g o f a more s p e c i f i c mechanism f o r p r i m a r y and s e c o n d a r y p r o p a r g y l i c a l c o h o l s was c o n f i r m e d i n a subsequent s t u d y o n t h e f a t h e a d minnow i n which 96-h LC50 v a l u e s were d e t e r m i n e d for a series of acetylenic alcohols. A l l p r i m a r y and s e c o n d a r y p r o p a r g y l i c a l c o h o l s were f o u n d t o produce e x c e s s t o x i c i t y , c o n s i s t e n t w i t h a p r o e l e c t r o p h i l e mechanism i n v o l v i n g t r a n s f o r m a t i o n by a l c o h o l dehydrogenase i n the f i s h t o the corresponding a,^-unsaturated p r o p a r g y l i c a l d e h y d e o r ketone e l e c t r o p h i l i c t o x i c a n t . F u r t h e r m o r e , t h e t o x i c i t i e s o f t e r t i a r y p r o p a r g y l i c a l c o h o l s , w h i c h c a n n o t undergo t h i s m e t a b o l i c t r a n s f o r m a t i o n , were v e r y c l o s e t o t h e QSAR p r e d i c t i o n s . P r i m a r y h o m o p r o p a r g l i c a l c o h o l s were a l s o f o u n d t o produce e x c e s s toxicity. A mechanism i n v o l v i n g t a u t o m e r i z a t i o n o f t h e a l d e h y d e m e t a b o l i t e t o t h e c o n j u g a t e d a l l e n e was p r o p o s e d . T h i s a l l e n e c a n a c t as a M i c h a e l - t y p e a c c e p t o r e l e c t r o p h i l e ( V e i t h , G.D.; L i p n i c k , R.L.; Russom, C.L. Xenobiotica, i n press). Local QSAR Models. F i s h t o x i c i t y s c r e e n i n g d a t a have a l s o been employed t o i n v e s t i g a t e t h e l i m i t a t i o n s o f " l o c a l " QSAR models ( F i g u r e 2) f o r p h e n o l s (102) and a n i l i n e s (203) u s i n g t h e s e same d a t a . Most o f t h e s e d a t a a r e from s t u d i e s c o n d u c t e d o r s p o n s o r e d b y t h e U.S. F i s h and W i l d l i f e S e r v i c e (USFWS). The S e r v i c e has s c r e e n e d thousands o f c h e m i c a l s f o r e f f e c t s u s i n g a wide range o f organisms and r o u t e s o f

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

384

PROBING BIOACTIVE MECHANISMS

Log T

e

F i g u r e 7. R e l a t i o n s h i p b e t w e e n e x c e s s t o x i c i t y (Tg) a n d c h r o n i c i t y r a t i o (R) f o r t h e t o x i c i t y o f 37 n o n e l e c t r o l y t e o r g a n i c c o m p o u n d s t o t h e f a t h e a d m i n n o w as f o l l o w s ( b y i n c r e a s i n g T g ) : 2,4,5-Trimethyloxazole (5), N , N - D i m e t h y l - p - t o l u i d i n e (6), 4 - M e t h y l o x a z o l e ( 7 ) , m-Bromobenzamide (8), 2 - F l u o r o t o l u e n e (9), n - B u t y l s u l f i d e (10), 1,2-Dichloropropane (11), 4,7-Dithiadecane (12), Naphthalene (13), 1,3-Dichloropropane (14), 4-Benzoylpyridine (15), 1,1,1,3,3,3-Hexafluoropropan-2-ol (16), t - B u t y l d i s u l f i d e ( 1 7 ) , Butan-2-one oxime ( 1 8 ) , s - T r i o x a n e ( 1 9 ) , F u r a n ( 2 0 ) , 2,4-Dichlorobenzamide (21), Pyrrole (22), Deca-1,9-diene (23), 2C y a n o p y r i d i n e ( 2 4 ) , 2-Adamantanone (25), H e x a n a l (26), Adamantane ( 2 7 ) , 1,4-Dicyanobutane ( 2 8 ) , C h l o r o m e t h y l s t y r e n e (29), 2 , 6 - D i p h e n y l p y r i d i n e ( 3 0 ) , 3 , 4 - D i c h l o r o b u t - l - e n e ( 3 1 ) , 2 , 4 , 5 - T r i m e t h o x y b e n z a l d e h y d e ( 3 2 ) , 4Nitrobenzamide ( 3 3 ) , Pentane-2,4-dione ( 3 4 ) , A l l y l cyanide ( 3 5 ) , P h e n y l disulfide (36), Benzaldehyde (37), 2,5-Diphenylfuran (38), a,a,aTrifluoro-m-tolualdehyde (39), Butanal (40), Pentafluorobenzaldehyde (41), a , a - D i c h l o r o - p - x y l e n e (42), 1,3-Dibromopropane (43), Acetaldehyde ( 4 4 ) , and C h l o r o a c e t o n i t r i l e ( 4 5 ) . ( F i g u r e adapted from data i n Tables 2 and 3 i n R e f . 88).

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

23.

UPNICK

Base-Line Toxicity Predicted by QSAR

385

a d m i n i s t r a t i o n (104). I n t h e e a r l y 1950s, Wood (98) t e s t e d 3,400 c h e m i c a l s f o r t o x i c i t y t o 3-4 s p e c i e s o f f r e s h w a t e r f i s h e s , f o l l o w e d by work o f H o l l i s and Lennon on t h e s e same o r g a n i s m s ( 9 9 ) , b o t h a t t h e Service's Laboratory i n Kearneysville, WV. The c h e m i c a l s were s u b s e q u e n t l y t r a n s f e r r e d t o a n o t h e r USFWS l a b o r a t o r y i n M i c h i g a n and u s e d as p a r t o f a s t u d y t o s e a r c h f o r a s e l e c t i v e l a m p r i c i d a l agent ( 2 0 0 ) . The most t o x i c c h e m i c a l s were t h e n s e n t t o t h e USFWS L a b o r a t o r y i n G a l v e s t o n , T e x a s , and were t e s t e d i n s e a r c h o f an agent s e l e c t i v e l y t o x i c t o t h e r e d t i d e d i n o f l a g e l l a t e ( 2 0 5 ) ; and s u b s e q u e n t l y t o t h e U n i v e r s i t y o f Idaho, where t h e y were t e s t e d f o r s e l e c t i v e t o x i c i t y t o v a r i o u s f i s h s p e c i e s ( 2 0 2 ) . The u s e o f c h e m i c a l s as p i s c i c i d a l a g e n t s has been r e v i e w e d i n a R u s s i a n p u b l i c a t i o n ( 2 0 6 ) . Conclusion QSAR models c a n be a v a l u a b l e means o f p r e d i c t i n g t h e t o x i c i t y o f untested n o n e l e c t r o l y t e organic chemicals. The models need t o be d e r i v e d from a s e r i e s o f c h e m i c a l s a c t i n g by a common m o l e c u l a r mechanism and encompass an adequate domain o f spanned s u b s t i t u e n t space i n t h e i r p h y s i c a l and c h e m i c a l p r o p e r t i e s . The a c u t e t o x i c i t y o f many classes of nonelectrolytes i s consistent with a narcosis or baseline t o x i c i t y mechanism. The a b i l i t y t o a p p l y s u c h models f o r p r e d i c t i v e purposes a l s o r e q u i r e s i n f o r m a t i o n t o suggest that the candidate c h e m i c a l a c t s b y t h e same mechanism. Acknowledgments The a u t h o r i s g r a t e f u l t o D r . James H. G i l f o r d , C h i e f , E n v i r o n m e n t a l Effects Branch, U.S. E n v i r o n m e n t a l Protection Agency forhis encouragement i n t h e p r e p a r a t i o n o f t h i s a r t i c l e . The a u t h o r w i s h e s t o t h a n k E l s e v i e r P u b l i s h i n g Company f o r k i n d l y p e r m i t t i n g t h e r e p r o d u c t i o n o f F i g u r e s from QSAR in Drug Design and Toxicology ( 6 4 ) .

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