The Elusive MetaboliteâThe Reactive Intermediate - American

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The Pesticide Chemist and Modern Toxicology Downloaded from pubs.acs.org by NORTH CAROLINA STATE UNIV on 11/07/18. For personal use only.

The Elusive Metabolite—The Reactive Intermediate JAMES R. GILLETTE Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, MD 20205

The ideal pesticide kills the target organism without affecting other organisms. But one of the major difficulties in interpreting toxicity data is that we seldom know why a given substance is more toxic in one animal species than in another species. Sometimes species differences in the response to a substance are related to differences in the affinity and number of receptor sites that combine with the substance. But other species differences are caused by the rates at which the substances are absorbed from administration sites, distributed to various body tissues, metabolized and eliminated from the body. Moreover, in recent years, it has become increasingly evident that biological responses to substances may be caused at least in part by metabolites of the substance. In some cases the response caused by the metabolite is similar to that of the parent substance but a l l too frequently the response caused by the metabolite is entirely different from the parent compound. Furthermore, toxic responses may be caused not only by chemically stable metabolites but also by metabolites that rapidly react irreversibly with various tissue components including proteins, lipids, and nucleic acids. The half-life of these substances can range from milliseconds to several hours. Thus some metabolites may have such short half-lives that they never leave the immediate environment of the enzymes that catalyze their formation, whereas other chemically reactive metabolites have sufficiently long half-lives that they leave the tissues in which they are formed, enter other tissues of the body and are excreted into urine. The d i f f e r e n t i a t i o n of the t o x i c p o t e n t i a l s of parent compounds from those of chemically s t a b l e metabolites i s r e l a t i v e l y simple. When a response depends on the r e v e r s i b l e binding of the drug or metabolite to receptor s i t e s and appears soon a f t e r the a d m i n i s t r a t i o n of drug, the i n t e n s i t y and d u r a t i o n of the response f r e q u e n t l y depends on the drug c o n c e n t r a t i o n i n blood. Studying the r e l a t i o n s h i p between the d u r a t i o n of a c t i o n of a drug and the c o n c e n t r a t i o n i n blood, however, w i l l f a i l when the response i s caused i n part by a metaboThis chapter not subject to U.S. copyright. Published 1981 American Chemical Society

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l i t e and t h e r a t e c o n s t a n t o f e l i m i n a t i o n o f t h e metabolite i s g r e a t e r than t h a t of the p a r e n t d r u g . Under t h e s e c o n d i t i o n s , the h a l f - l i f e of the m e t a b o l i t e d u r i n g the t e r m i n a l p h a s e w i l l a p p e a r t o be t h e same as t h a t o f t h e d r u g and t h u s t h e d u r a t i o n o f a c t i o n may a p p e a r t o be a p p r o x i m a t e l y r e l a t e d t o t h e c o n c e n t r a t i o n o f t h e p a r e n t d r u g e v e n when i t i s c a u s e d s o l e l y by t h e m e t a b o l i t e . A b e t t e r strategy to e l u c i d a t e t h e t o x i c o l o g i c a l e f f e c t s o f c h e m i c a l l y s t a b l e and l o n g - l i v e d c h e m i c a l l y r e a c t i v e m e t a b o l i t e s i s t o i s o l a t e and i d e n t i f y t h e m e t a b o l i t e s , s y n t h e s i z e them, and t e s t them f o r their toxic activities. Standard pharmacokinetic concepts may t h e n be a p p l i e d t o e v a l u a t e t h e r e l a t i v e c o n t r i b u t i o n s o f t h e p a r e n t compound and t h e m e t a b o l i t e s i n t h e m a n i f e s t a t i o n of the t o x i c i t y . T h e s e s t r a t e g i e s w i l l f a i l , however, when t h e t o x i c i t y i s c a u s e d by s h o r t - l i v e d c h e m i c a l l y r e a c t i v e m e t a b o l i t e s . Such m e t a b o l i t e s a r e n o t e a s i l y i s o l a t e d and t h u s t h e i r i d e n t i t y must be i n f e r r e d f r o m i n d i r e c t e v i d e n c e b a s e d on t h e i r u l t i m a t e decomposition products. Even i f the c h e m i c a l l y r e a c t i v e m e t a b o l i t e s were i d e n t i f i e d t h e y w o u l d n o t be e a s i l y synthesized or p u r i f i e d . Moreover, t h e i r t o x i c p o t e n t i a l i s not e a s i l y s t u d i e d b e c a u s e t h e y w o u l d be i n a c t i v a t e d d u r i n g t h e i r p a s s a g e from the s i t e s of a d m i n i s t r a t i o n to t h e i r t a r g e t o r g a n s . C l e a r l y a d i f f e r e n t s t r a t e g y must be e m p l o y e d t o d e t e r m i n e w h i c h chemi c a l l y r e a c t i v e m e t a b o l i t e s a r e t o x i c and w h i c h a r e i n n o c u o u s . S e v e r a l y e a r s ago my c o l l e a g u e s and I d e v i s e d a s t r a t e g y t o d e t e r m i n e w h e t h e r a g i v e n t o x i c i t y i s c a u s e d by a c h e m i c a l l y reactive metabolite. I n d e v e l o p i n g t h e a p p r o a c h (1,2), we considered that chemically r e a c t i v e metabolites conceivably c o u l d c a u s e t o x i c r e a c t i o n s , s u c h as a c e l l u l a r n e c r o s i s , t h r o u g h s e v e r a l d i f f e r e n t mechanisms ( F i g . l ) . Conceivably, the t a r g e t of the c h e m i c a l l y r e a c t i v e m e t a b o l i t e c o u l d be an i n t r a c e l l u l a r enzyme o r i t s s u b s t r a t e s r e q u i r e d f o r the f u n c t i o n o f c e l l s . I t c o u l d be a p h o s p h o l i p i d i n c e l l u l a r membranes, w h i c h c o n t r o l t h e i n t r a c e l l u l a r c o m p a r t m e n t a l i z a t i o n of i n t r a c e l l u l a r components. I t c o u l d be p a r t o f t h e p r o t e i n s y n t h e s i s machinery r e q u i r e d f o r the normal replacement of i n t r a c e l l u l a r enzymes. I t c o u l d a l s o be DNA r e q u i r e d f o r c e l l u l a r replication. We a l s o e n v i s i o n e d t h e p o s s i b i l i t y t h a t t h e m a n i f e s t a t i o n of the t o x i c i t y might not occur u n l e s s s e v e r a l o f t h e s e t a r g e t s were i m p a i r e d simultaneously. I t o c c u r r e d t o us t h a t i n c a u s i n g a l t e r a t i o n s o f t h e t a r g e t substances, the c h e m i c a l l y r e a c t i v e m e t a b o l i t e might a l t e r the t a r g e t s u b s t a n c e s by c o m b i n i n g c o v a l e n t l y w i t h them. I t was a l s o p l a u s i b l e , however, t h a t t h e t o x i c r e s p o n s e m i g h t be c a u s e d by m e c h a n i s m s i n w h i c h t h e c h e m i c a l l y r e a c t i v e m e t a b o l i t e i s n o t c o v a l e n t l y bound t o t h e t a r g e t s u b s t a n c e . The chemically r e a c t i v e m e t a b o l i t e m i g h t r e a c t w i t h a l i p i d o r DNA t o f o r m r e a c t i v e e n d o g e n o u s components and t h e r e b y c a u s e t h e t o x i c i t y ; f o r example, the r e a c t i o n o f t r i c h l o r o m e t h y l f r e e r a d i c a l w i t h

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GILLETTE

Elusive

Metabolite—Reactive

Parent Foreign Compound

Reactire Metabolite!

}

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Reactive Metabolites

Figure 1.

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Intermediate

Ccry aient Binding t o Proteins Including Enzymes

Toxicityt

Codaient Binding t o RHA and DHA

Toxicityt

CoTalent Binding t o Lipide

Toxicityt

Inert Metabolite + Reactive Endogenous Component (Lipid or DHA Free Radicale)

Toxicityt

Inert Metabolite + Superoxide, >^ Hydrogen Peroxide or Hydroxy! Radical

Covalent Binding to Snail Molecular Weight Substances (ATP, UDPO, etc.)

Toxicityt

•

Toxicityt

Postulated mechanisms of toxicity by chemically reactive metabolites

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l i p i d t o form c h l o r o f o r m and l i p i d f r e e r a d i c a l s has been s u g g e s t e d as an i n i t i a l s t e p i n t h e l i v e r n e c r o s i s c a u s e d by carbon t e t r a c h l o r i d e (3). A chemically r e a c t i v e metabolite may r e a c t w i t h o x y g e n To f o r m s u p e r o x i d e , h y d r o g e n p e r o x i d e or hydroxyl f r e e r a d i c a l s which i n t u r n causes the t o x i c i t y A t t h e t i m e we were d e v e l o p i n g o u r a p p r o a c h , we were w e l l aware o f t h e many s t u d i e s by o n c o l o g i s t s i n d i c a t i n g r e l a t i o n s h i p s between c a r c i n o g e n e s i s and t h e f o r m a t i o n o f t h e c h e m i c a l l y r e a c t i v e m e t a b o l i t e s o f f o r e i g n compounds (5,6,7,8) These s t u d i e s profoundly i n f l u e n c e d our thoughts. I t was e v i d e n t t h a t most c h e m i c a l l y r e a c t i v e m e t a b o l i t e s do n o t r e a c t with a s i n g l e k i n d of macromolecule, but i n s t e a d r e a c t w i t h many t i s s u e components i n c l u d i n g p r o t e i n s , l i p i d , n u c l e i c a c i d s , g l y c o g e n and m i c r o m o l e c u l a r s u b s t a n c e s , such as ATP, NADPH, NADH and UDPG. I t was a l s o e v i d e n t t h a t t h e rates of r e a c t i o n of a given metabolite with the various n u c l e o p h i l e s i n c e l l s d e p e n d on s e v e r a l f a c t o r s . F o r example, the r a t e s o f r e a c t i o n w i t h t h i o l groups d i f f e r markedly from t h e r a t e s o f r e a c t i o n w i t h amino g r o u p s o f p r o t e i n s and oxygen o r n i t r o g e n groups o f the n u c l e i c a c i d s . Hence a m u l t i p l i c i t y o f d i f f e r e n t r e a c t i o n p r o d u c t s may o c c u r . On the o t h e r hand, r a t h e r s t a b l e c h e m i c a l l y r e a c t i v e m e t a b o l i t e s may c o m b i n e r e v e r s i b l y w i t h c e r t a i n s i t e s o f some p r o t e i n s b e f o r e t h e c o m p l e x r e a r r a n g e s t o c o v a l e n t l y bound m a t e r i a l . I n t h i s c a s e , low c o n c e n t r a t i o n s o f t h e r e a c t i v e m e t a b o l i t e may combine w i t h r e l a t i v e l y few k i n d s o f m a c r o m o l e c u l e s . I n d e e d , t h e i n h i b i t i o n o f p s e u d o c h o l i n e s t e r a s e s by p h o s p h o r u s i n s e c t i c i d e s i s a n example o f t h i s k i n d o f m e c h a n i s m . F o r t h e s e r e a s o n s , a c h e m i c a l l y r e a c t i v e m e t a b o l i t e may combine p r e f e r e n t i a l l y with c e r t a i n c e l l u l a r p r o t e i n s because they c o n t a i n a n u n u s u a l l y l a r g e number o f n u c l e o p h i l i c g r o u p s o n the s u r f a c e o f the p r o t e i n o r because t h e p r o t e i n has a h i g h a f f i n i t y f o r the r e a c t i v e metabolite. Because d i f f e r e n t c h e m i c a l l y r e a c t i v e m e t a b o l i t e s react with various tissue nucleophiles at r e l a t i v e l y d i f f e r e n t r a t e s , i t seemed l i k e l y t o u s t h a t m e a s u r i n g t h e t o t a l c o v a l e n t b i n d i n g o f r e a c t i v e m e t a b o l i t e s t o p r o t e i n s would n o t p r o v i d e a r e l i a b l e estimate of the r e l a t i v e t o x i c i t y o f the chemically reactive metabolites. I n d e e d i t seemed e n t i r e l y p o s s i b l e t h a t a chemically reactive metabolite could react extensively with p r o t e i n a n d s t i l l be n o n t o x i c . M o r e o v e r , i t a l s o seemed p o s s i b l e t h a t a t o x i c a n t m i g h t be c o n v e r t e d t o a c h e m i c a l l y react i v e m e t a b o l i t e which combined w i t h p r o t e i n even though t h e t o x i c i t y i s c a u s e d d i r e c t l y by t h e p a r e n t s u b s t a n c e . I t o c c u r r e d t o u s , h o w e v e r , t h a t we m i g h t be a b l e t o d e t e r m i n e w h e t h e r a t o x i c i t y was c a u s e d by c h e m i c a l l y r e a c t i v e m e t a b o l i t e s by s t u d y i n g t h e e f f e c t s o f v a r i o u s i n d u c e r s a n d i n h i b i t o r s of the metabolism o f the t o x i c a n t . According to our view, t h e c o v a l e n t b i n d i n g o f t h e r e a c t i v e m e t a b o l i t e t o p r o t e i n w o u l d be a p p r o x i m a t e l y p r o p o r t i o n a l t o t h e a r e a u n d e r

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the c e l l u l a r c o n c e n t r a t i o n c u r v e o f t h e c h e m i c a l l y r e a c t i v e m e t a b o l i t e a n d t h e r e f o r e , a n i n d i r e c t m e a s u r e o f t h e amount o f r e a c t i v e m e t a b o l i t e i n c o n t a c t w i t h t h e t a r g e t component in cells. Thus, any t r e a t m e n t t h a t r e s u l t s i n a change i n the a r e a under t h e curve o f the c h e m i c a l l y r e a c t i v e m e t a b o l i t e would cause p a r a l l e l changes i n both t h e c o v a l e n t b i n d i n g o f the m e t a b o l i t e t o p r o t e i n and t h e s e v e r i t y o f t h e t o x i c i t y when t h e t o x i c i t y i s c a u s e d by t h e c h e m i c a l l y r e a c t i v e m e t a b o l i t e o r a m e t a b o l i t e d e r i v e d from i t . Moreover, t h e c o r r e l a t i o n s h o u l d o c c u r e v e n when t h e c h e m i c a l l y r e a c t i v e m e t a b o l i t e d o e s n o t c a u s e t h e t o x i c i t y by c o v a l e n t b i n d i n g t o a n y i n t r a c e l l u l a r component. When t h e t o x i c i t y r e s u l t s f r o m c o v a l e n t b i n d i n g o f t h e m e t a b o l i t e t h e a p p r o a c h may be e x p r e s s e d mathematically. T h e amount o f m e t a b o l i t e t h a t c o m b i n e s w i t h a t a r g e t s u b s t a n c e may be e x p r e s s e d a s t h e d o s e t i m e s t h e f r a c t i o n o f t h e dose that i s c o n v e r t e d t o a c h e m i c a l l y r e a c t i v e m e t a b o l i t e ( R a t i o A) times the f r a c t i o n o f the c h e m i c a l l y r e a c t i v e m e t a b o l i t e t h a t becomes c o v a l e n t l y bound t o t h e target substance (Ratio B). S i m i l a r l y t h e amount o f m e t a b o l i t e t h a t u l t i m a t e l y becomes c o v a l e n t l y bound t o p r o t e i n i n t h e t a r g e t t i s s u e may a l s o be e x p r e s s e d a s t h e f r a c t i o n o f t h e d o s e o f t h e t o x i c a n t t h a t becomes c o v a l e n t l y bound t o p r o t e i n and t h i s f r a c t i o n may be e x p r e s s e d a s R a t i o A t i m e s t h e f r a c t i o n o f t h e r e a c t i v e m e t a b o l i t e t h a t becomes c o v a l e n t l y bound t o p r o t e i n ( R a t i o B ) . Thus, T a r g e t - M e t a b o l i t e - Dose A Β P r o t e i n - M e t a b o l i t e = Dose A B' I t f o l l o w s , t h e r e f o r e t h a t any treatment t h a t changes R a t i o A or both R a t i o Β and R a t i o Β w i t h o u t s u b s t a n t i a l l y changing the r e l a t i v e r a t e s o f t h e r e a c t i o n s o f t h e m e t a b o l i t e s with p r o t e i n and t h e t a r g e t s u b s t a n c e w i l l r e s u l t i n p a r a l l e l changes i n t h e s e v e r i t y o f t h e t o x i c i t y and t h e c o v a l e n t bind i n g t o p r o t e i n e v e n when t h e t a r g e t s u b s t a n c e i s n o t a p r o t e i n . Thus, d e t e r m i n i n g t h e e f f e c t s o f v a r i o u s treatments that a r e known t o a l t e r t h e m e t a b o l i s m o f t h e t o x i c a n t o r t h e i n a c t i v a t i o n o f t h e m e t a b o l i t e s i s u s e f u l i n d e t e r m i n i n g whether a t o x i c i t y i s m e d i a t e d by a c h e m i c a l l y r e a c t i v e m e t a b o l i t e . The c o n c e p t may a l s o be e x p a n d e d t o i n c l u d e s i t u a t i o n s i n which the chemically r e a c t i v e metabolite that reacts with p r o t e i n s a l s o i s converted t o another m e t a b o l i t e that causes toxicity. I n t h i s s i t u a t i o n any t r e a t m e n t t h a t c a u s e s a change i n t h e f r a c t i o n o f t h e dose t h a t i s c o n v e r t e d t o a n o t h e r m e t a b o l i t e w i l l cause p a r a l l e l changes i n t h e c o v a l e n t binding of t h e m e t a b o l i t e t o p r o t e i n and t h e s e v e r i t y o f t h e t o x i c i t y . But a t r e a t m e n t t h a t p r e f e r e n t i a l l y a l t e r s t h e c o n v e r s i o n of the chemically r e a c t i v e metabolite t o the t o x i c metabolite would cause i n v e r s e l y r e l a t e d changes i n t h e magnitude o f t h e c o v a l e n t b i n d i n g and t h e s e v e r i t y o f t h e t o x i c i t y . I n a d d i t i o n t o v a r i o u s t r e a t m e n t s t h a t a l t e r enzyme a c t i v i t i e s , c h a n g e s i n R a t i o s A, Β a n d B may a l s o o c c u r b y changes i n t h e s i z e o f t h e dose o f t h e t o x i c a n t . A t low 1

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doses the r a t e s of conversion of the parent compound to i t s various metabolites i n c l u d i n g the chemically r e a c t i v e metabo l i t e and the r a t e s of d i s p o s i t i o n of the chemically r e a c t i v e metabolite w i l l be f i r s t order. Under these c o n d i t i o n s the values of the Ratios A, Β and B w i l l be independent of the dose. But as the dose i s increased, the maximum c o n c e n t r a t i o n of the parent compound reached i n the organ of e l i m i n a t i o n may be s u f f i c i e n t to saturate one or more of the enzymes that c a t a l y z e i t s metabolism, and thereby R a t i o A may be changed. If the enzyme that has the lowest K c a t a l y z e s the formation of the chemically r e a c t i v e metabolite Ratio A w i l l be decreased. But i f the enzyme having the lowest c a t a l y z e s the formation of an innocuous metabolite Ratio A w i l l be i n c r e a s e d . More over, increases i n the dose of the parent compound w i l l lead to increases i n the amount of chemically r e a c t i v e metabolite formed, and i n turn may lead to the d e p l e t i o n of i n t r a c e l l u l a r n u c l e o p h i l e s , such as g l u t a t h i o n e ; thus increases i n the dose may lead to an increase i n Ratios Β and B . Ratio A i n the target t i s s u e may a l s o be changed by an a l t e r a t i o n i n the a c t i v i t y of an enzyme i n a t i s s u e other than the i n i t i a l target t i s s u e and i n some cases by changing the route of a d m i n i s t r a t i o n . An understanding of the k i n e t i c s of these e f f e c t s i s e s p e c i a l l y important f o r they can account i n part f o r a s h i f t of the t o x i c i t y from one organ to another caused by various treatments ( 9 ) . Under these c o n d i t i o n s , Ratio A would be the amount of substance converted to the chemically r e a c t i v e metabolite i n the target t i s s u e d i v i d e d by the sum of amounts of the substance metabolized and other wise e l i m i n a t e d i n the t a r g e t t i s s u e and the other t i s s u e s of the body. With these c o n s i d e r a t i o n s i n mind, we devised the f o l l o w ing sequence of i n v i v o and i n v i t r o experiments by which we determine whether a given t o x i c i t y i s caused by a chemically r e a c t i v e metabolite. 1) Determine whether the substance causes t o x i c i t i e s i n v a r i o u s species and s t r a i n s of animals. Obviously one cannot study the mechanism of a t o x i c i t y i n an animal species when the t o x i c i t y does not occur i n that s p e c i e s . S u r p r i s i n g l y , however, many i n v e s t i g a t o r s spend considerable e f f o r t i n e l u c i d a t i n g the pharmacokinetics and the pattern of the metabo l i s m of a substance, i n a given species before they demonstrate that the substance i s t o x i c i n that s p e c i e s . By f i r s t c a r r y ing out t o x i c i t y s t u d i e s i n d i f f e r e n t animal species the i n v e s t i g a t o r can choose the species with which f u r t h e r s t u d i e s may be c a r r i e d out. 2) Determine the dose-response r e l a t i o n s h i p s of the substance and the t o x i c i t y i n the d i f f e r e n t animal s p e c i e s . This i s a n a t u r a l consequence of step 1, since the i n v e s t i g a t o r should administer s e v e r a l d i f f e r e n t doses of the substance i n e v a l u a t i n g species d i f f e r e n c e s i n the incidence and s e v e r i t y of the t o x i c i t y . f

m

f

14.

GILLETTE

Elusive


Intermediate

223

3) D e v e l o p a n a l y t i c a l methods f o r t h e a s s a y o f t h e s u b stance and i t s major m e t a b o l i t e s formed i n t h e a n i m a l * 4) When t h e t o x i c i t y i s m a n i f e s t e d by a s i n g l e d o s e o f t h e s u b s t a n c e , s t u d y w h e t h e r p r e t r e a t m e n t s t h a t a r e known t o a l t e r t h e r a t e o r p a t t e r n o f m e t a b o l i s m o f f o r e i g n compounds w i l l a l t e r the incidence or s e v e r i t y of the t o x i c i t y . 5) Compare t h e e f f e c t s o f t h e p r e t r e a t m e n t s o n t h e t o t a l body c l e a r a n c e a n d t h e p a t t e r n o f m e t a b o l i t e s o f t h e t o x i c a n t . F r e q u e n t l y , such s t u d i e s e l u c i d a t e whether t h e t o x i c i t y i s c a u s e d b y t h e p a r e n t compound o r b y a m e t a b o l i t e . 6) D e t e r m i n e w h e t h e r s u b s t a n c e s r a d i o l a b e l e d a t m e t a b o l i c a l l y s t a b l e p o s i t i o n s o f t h e s u b s t a n c e become c o v a l e n t l y bound t o compounds i n t h e t a r g e t t i s s u e s . Subtoxic as w e l l as t o x i c d o s e s s h o u l d be s t u d i e d i n o r d e r t o d e t e r m i n e w h e t h e r t h e c o v a l e n t b i n d i n g f o l l o w s f i r s t order k i n e t i c s o r whether t h e r e a r e t h r e s h o l d doses below which c o v a l e n t b i n d i n g i s u n important. 7) D e t e r m i n e w h e t h e r t h e e f f e c t s o f t h e p r e t r e a t m e n t s t h a t a l t e r t h e p a t t e r n and r a t e s o f metabolism o f t h e t o x i c a n t cause p a r a l l e l c h a n g e s i n t h e amount o f r a d i o l a b e l c o v a l e n t l y bound t o components i n t h e t a r g e t a n d o t h e r t i s s u e s . P a r a l l e l c h a n g e s i n d i c a t e t h a t t h e t o x i c i t y and t h e c o v a l e n t b i n d i n g a r e caused by a common i n t e r m e d i a t e a n d may be c a u s e d b y t h e same i n t e r mediate. I n v e r s e l y r e l a t e d c h a n g e s s u g g e s t t h a t t h e two phenomena a r e c a u s e d by two i n t e r m e d i a t e s t h a t a r e f o r m e d f r o m a common i n t e r m e d i a t e . 8) D e t e r m i n e w h e t h e r t h e v a r i o u s t r e a t m e n t s a l t e r t h e i n v i t r o a c t i v i t y o f enzymes t h a t c a t a l y z e t h e f o r m a t i o n o f t h e c h e m i c a l l y r e a c t i v e intermediate not only i n the target organ but a l s o i n o t h e r t i s s u e s ( l i v e r f o r example) t h a t m e t a b o l i z e t h e f o r e i g n compound. The as w e l l as the V o f t h e enzymes s h o u l d be c a l c u l a t e d i n o r d e r t o e s t i m a t e t h e i n t r i n s i c c l e a r a n c e s o f t h e enzymes ( i . e . V / K ) i n t h e d i f f e r e n t t i s s u e s and t o a s s e s s t h e p o s s i b i l i t y t h a t t h e c o n c e n t r a t i o n o f t h e s u b s t a n c e m i g h t r e a c h l e v e l s i n t h e body t h a t w o u l d r e s u l t i n nonlinear kinetics. Such s t u d i e s i n c o m b i n a t i o n w i t h s t u d i e s i n v i v o f r e q u e n t l y a r e u s e f u l i n a s s e s s i n g whether the r e a c t i v e metabolite i s s u f f i c i e n t l y s t a b l e t o escape the organ o f f o r m a t i o n a n d be c a r r i e d t o o t h e r t a r g e t o r g a n s . 9) I d e n t i f y t h e d e c o m p o s i t i o n p r o d u c t s f o r m e d f r o m t h e chemi c a l l y reactive metabolites. S t u d i e s on t h e e f f e c t s o f v a r i o u s n u c l e o p h i l e s s u c h a s g l u t a t h i o n e , v a r i o u s amino a c i d s a n d p u r i n e and p y r i m i d i n e b a s e s f r e q u e n t l y p r o v i d e c l u e s t o t h e t y p e s m

m

of

a

x

m

adducts formed from t h e c h e m i c a l l y r e a c t i v e m e t a b o l i t e s . 10) O b t a i n s u p p o r t i v e e v i d e n c e t h a t t h e t o x i c i t y i s m e d i a t e d by a c h e m i c a l l y r e a c t i v e m e t a b o l i t e . F o r example, when a c h e m i c a l l y r e a c t i v e m e t a b o l i t e r e a c t s w i t h g l u t a t h i o n e to form a conjugate, the c o n c e n t r a t i o n o f g l u t a t h i o n e i n the target t i s s u e i s frequently decreased. The s e v e r i t y o f t h e t o x i c i t y thus i s f r e q u e n t l y i n c r e a s e d by t h e a d m i n i s t r a t i o n of s u b s t a n c e s (such as d i e t h y l m a l e a t e ) t h a t a l s o r e a c t w i t h

224


g l u t a t h i o n e and decreased by the a d m i n i s t r a t i o n of a l t e r n a t i v e n u c l e o p h i l e s or precursors of g l u t a t h i o n e . Such s t u d i e s thus lead to the discovery of antidotes to acute t o x i c i t i e s caused by chemically r e a c t i v e metabolites. With t h i s approach we and others have discovered that s e v e r a l commonly used drugs can cause t i s s u e damage through the formation of metabolites (Table 1). In a d d i t i o n the s t u d i e s on the e f f e c t s of inducers, i n h i b i t o r s and p o t e n t i a l n u c l e o p h i l e s on the covalent binding of chemically r e a c t i v e metabolites formed i n v i t r o have helped us to understand the c h a r a c t e r i s t i c s and p r o p e r t i e s of the chemically r e a c t i v e metabolites and the enzymes that c a t a l y z e t h e i r formation and inactivation. Our s t u d i e s on the metabolism of phenacetin and acetaminophen i l l u s t r a t e how we have used t h i s coordinated approach i n studying t o x i c i t i e s caused by chemically r e a c t i v e metabolites. I t i s w e l l known that l a r g e overdoses of acetaminophen cause f a t a l hepatic n e c r o s i s not only i n man (10) but a l s o i n s e v e r a l l a b o r a t o r y animal species such as r a t s (11,12), mice (12) and hamsters (13>14)· There i s a marked species d i f f e r e n c e i n the s e n s i t i v i t y of the v a r i o u s species to the drug (Table 2). In hamsters, n e c r o s i s occurs i n most of the animals even at doses as low as 150 mg/kg, whereas i n some s t r a i n s of r a t s n e c r o s i s occurs i n l e s s than 10% of the animals even at doses as high as 1.5 g/kg (13)· Acetaminophen administered to animals i s excreted mainly as i t s glucuronide and i t s s u l f a t e conjugate (15), but a small amount of the drug i s excreted as i t s mercapturic a c i d and c y s t e i n e d e r i v a t i v e s i n a l l animals studied i n c l u d i n g man (16) ( F i g . 2 ) . Studies on the covalent binding of the r a d i o l a b e l to l i v e r p r o t e i n a f t e r the a d m i n i s t r a t i o n of v a r i o u s doses of r a d i o l a b e l e d acetaminophen to mice revealed that only n e g l i g i b l e amounts of the drug were c o v a l e n t l y bound at doses below 100 mg/kg (17)· At higher doses, however, considerable r a d i o l a b e l was c o v a l e n t l y bound to l i v e r p r o t e i n . Moreover, the covalent binding appeared to be n e g l i g i b l e u n t i l the l i v e r was depleted of g l u t a t h i o n e . Since acetaminophen i s chemi c a l l y i n e r t , these f i n d i n g s thus i n d i c a t e d that i t was converted to a chemically r e a c t i v e metabolite i n mice. They f u r t h e r suggested that at low doses of the drug, v i r t u a l l y a l l of the metabolite i s converted to a g l u t a t h i o n e conjugate that i s u l t i m a t e l y excreted as a mercapturic a c i d . At high doses of the drug, the g l u t a t h i o n e i n l i v e r i s decreased to such an extent that the r e a c t i v e metabolite can no longer be completely i n a c t i v a t e d by g l u t a t h i o n e and thus a p o r t i o n of i t becomes c o v a l e n t l y bound to l i v e r p r o t e i n s . In accord with t h i s view, the p r o p o r t i o n of the dose of acetaminophen that i s excreted as the mercapturic a c i d i s about 10% when low doses of the drugs are administered to mice and i t decreases as the dose i s increased (18).

GILLETTE

TABLE 1

Elusive Metabolite—Reactive

Intermediate

225

Examples of the formation of c h e m i c a l l y r e a c t i v e metabolites

Compound

Tissue binding*

Bromobenzene (54)

H,L,K

Phenacetin (55) Acetaminophen (56,20,

H H

Furosemide (57) Ipomeanol (55) Various furans (59) I s o n i a z i d (60) I p r o n i a z i d (61) Carbon t e t r a c h l o r i d e (3,62,63,64) Chloroform (65) Chloramphenicol (66) N i t r o f u r a n t o i n (67) Benzene (68,69)

H L,H,K H,L,K H H H,K H Η,ΒΜ L L,BM

Pathway Intermediate(s)

Bromobenzene3,4-epoxide (?) Acetaminophen N-Acetimidoquinone ( ? ) Furosemide epoxide ( ? ) (?) (?) A c e t y l hydrazine Isopropyl hydrazine Trichloromethyl free radical Phosgene R-oxalyl chloride Reduction product (?)

*H • L i v e r , L = Lung, Κ = Kidney, BM = Bone marrow

226


TABLE 2

L i v e r n e c r o s i s caused by

Dose (mg/kg)

150

Mice

Incidence (%) Hamsters

0

Rats

0

200

20

300

22

375

46

425

-

500

89

100 7

750

99

1000

-

1500

acetaminophen

6

-

-

0

2 6

Data taken from M i t c h e l l et a l . (56) and P o t t e r et a l . (70). ~

14.

GILLETTE

Elusive


Intermediate

ACETAMINOPHEN

GLUCURONIDE

MACROMOLECULES

Figure 2. Principle pathways of acetaminophen metabolism

227


228

As the dose of acetaminophen was increased, the incidence and s e v e r i t y of the l i v e r n e c r o s i s i n mice was increased (12)· However, an increase i n t o x i c i t y would be expected to occur regardless of the mechanism of t o x i c i t y . Thus, the apparent c o r r e l a t i o n between the increase i n covalent binding and the incidence of t o x i c i t y based s o l e l y on changes i n the dose (12,17) i s only t r i v i a l and does not i n d i c a t e whether the t o x i c i t y i s caused by the parent compound, the chemically r e a c t i v e metabolite or some other metabolite. In order to determine whether the t o x i c i t y i s caused by a chemically r e a c t i v e metabolite the animals must be t r e a t e d with substances that would a l t e r e i t h e r Ratio A or Ratio B. Since the r e a c t i v e metabolite p r e f e r e n t i a l l y combines with g l u t a t h i o n e (17) the d e p l e t i o n of l i v e r g l u t a t h i o n e by other substances that react with g l u t a t h i o n e should increase the covalent binding of the r e a c t i v e metabolite by i n c r e a s i n g Ratio B, whereas the a d m i n i s t r a t i o n of c y s t e i n e should decrease i t . In accord with t h i s view d i e t h y l maleate, which decreases the concentration of g l u t a t h i o n e i n l i v e r but does not cause l i v e r n e c r o s i s (19), not only increases the covalent binding of the reactivêTmetabolite of acetaminophen, but a l s o increases the incidence and s e v e r i t y of the l i v e r n e c r o s i s i n mice (17). On the other hand, treatment of mice with c y s t e i n e decreases the covalent binding of the r e a c t i v e metabolite and decreases the incidence and s e v e r i t y of the l i v e r n e c r o s i s (17). Pretreatment of mice with phénobarbital increases the a c t i v i t y of the enzyme that c a t a l y z e s the formation of the r e a c t i v e metabolite and thus a c c e l e r a t e s the d e p l e t i o n of hepatic g l u t a t h i o n e (17), but apparently does not a f f e c t the enzymes that c a t a l y z e the formation of the s u l f a t e or the glucuronide conjugates because i t does not a l t e r the b i o l o g i c a l h a l f - l i f e of the drug i n mice (12)· Thus, pretreatment of mice with phénobarbital increases the p r o p o r t i o n of the dose of acetaminophen that becomes c o v a l e n t l y bound to l i v e r p r o t e i n by i n c r e a s i n g Ratio A and i n c r e a s e s the incidence and s e v e r i t y of the l i v e r n e c r o s i s (12,20)· Studies with l i v e r microsomes i n d i c a t e d that the f o r mation of the chemically r e a c t i v e metabolite, as measured by covalent binding of r a d i o l a b e l e d acetaminophen to microsomal p r o t e i n , i s c a t a l y z e d by a cytochrome P-450 enzyme i n l i v e r microsomes (21). They a l s o showed species d i f f e r e n c e s i n the k i n e t i c s f o r the formation of the r e a c t i v e metabolite. With l i v e r microsomes from mice the apparent maximal v e l o c i t y f o r the r e a c t i o n ( V ) was about 0.18 nmoles bound/mg p r o t e i n / min and the apparent was about 0.36 mM acetaminophen, whereas with l i v e r microsomes from r a t s , the apparent V was 0.07 nmoles/mg protein/min and the apparent 1^ was 14.8 mM acetaminophen. Since the i n t r i n s i c clearance of a s u b s t r a t e by an enzyme i n the body should be ν ^ χ / ζ Κ ^ + S ) , these f i n d ings are i n accord with the view that the r a t e of formation m a x

m a x

14.

GILLETTE

Elusive


Intermediate

of the r e a c t i v e metabolite would be slower i n r a t s than i n mice not only because the i s lower i n r a t s , but a l s o because the K i s higher. The a d d i t i o n of g l u t a t h i o n e to the i n c u b a t i o n mixtures i n the presence and absence of the s o l u b l e f r a c t i o n of l i v e r i n h i b i t e d the covalent binding of the r e a c t i v e metabolite to p r o t e i n , but r e s u l t e d i n the formation of an acetaminopheng l u t a t h i o n e conjugate (21,22,23,24). The f i n d i n g that the cov a l e n t binding was blocked at a lower g l u t a t h i o n e concent r a t i o n i n the presence of the s o l u b l e f r a c t i o n than i n i t s absence l e d to the c o n c l u s i o n that the formation of the g l u t a t h i o n e conjugate was c a t a l y z e d by one or more of the g l u t a t h i o n e t r a n s f e r a s e s i n l i v e r even though the conjugate can be formed nonenzymatically. Strangely, the sum of the covalent binding and the g l u t a t h i o n e conjugate a l s o increased (20,25) as d i d the r a t e of disappearance of acetaminophen (25) as the g l u t a t h i o n e c o n c e n t r a t i o n was i n c r e a s e d . I t , t h e r e f o r e , seems p o s s i b l e that a part i s reduced back to acetaminophen and that g l u t a t h i o n e prevents t h i s r e d u c t i o n by the formation of the conjugate. In accord with t h i s view, a s c o r b i c a c i d i n h i b i t s the covalent binding of the acetaminophen metabolite to p r o t e i n (26) and g l u t a t h i o n e decreases r a t h e r than increases acetaminophen dependent NADPH o x i d a t i o n by l i v e r microsomes (27)· m

Thus, the chemically r e a c t i v e metabolite appeared to be a s h o r t - l i v e d substance that reacts with g l u t a t h i o n e and i s e a s i l y r e d u c i b l e by a s c o r b i c a c i d . At f i r s t , we suggested that the metabolite that caused the l i v e r n e c r o s i s might be N-hydroxyacetaminophen (20,212,23,24-) . T h i s hypothesis was based p r i m a r i l y on the f i n d i n g of Calder et_ a l . that Nacetylimidoquinone (N-acetyl-p-benzoquinoneimine) was an e l e c t r o p h i l i c compound which could be formed from N-hydroxyphenacetin under a c i d i c c o n d i t i o n s (28). Thus, i t seemed p o s s i b l e that l i v e r microsomes might convert acetaminophen to N-hydroxyacetaminophen which i n t u r n undergoes spontaneous dehydration to the N-acetylimidoquinone. In support of t h i s hypothesis i t was shown that the acetaminophen analogs p - c h l o r o a c e t a n i l i d e (29,30) and phenacetin (31) were N-hydroxylated and the treatments of animals which a l t e r e d the microsomal N-hydroxylation of these analogs caused s i m i l a r changes i n the r a t e of formation of the e l e c t r o p h i l i c metabolite of acetaminophen by l i v e r microsomes. N-Hydroxyacetaminophen was r e c e n t l y synthesized and i t s chemical p r o p e r t i e s examined (32,33)· In aqueous s o l u t i o n s the proposed metabolite was unstable and presumably dehydrated to the e l e c t r o p h i l e N-acetylimidoquinone with a h a l f - l i f e of approximately 15 min. When i n j e c t e d i n t o mice the compound decreased the g l u t a t h i o n e c o n c n t r a t i o n i n l i v e r and was hepatotoxic ( 3 2 ) .

229

230


Recently, McMurtry et_ a l . showed acetaminophen i n F i s c h e r r a t s becomes c o v a l e n t l y bound to kidney as w e l l as to l i v e r (34). However, the chemically r e a c t i v e metabolite i n kidney appears to be produced i n the kidney r a t h e r than the l i v e r s i n c e 3-methylcholanthrene pretreatment increased the covalent binding i n the l i v e r but not the kidney. Thus i t seemed l i k e l y that the chemically r e a c t i v e metabolite of acetaminophen formed i n the l i v e r has too short a h a l f - l i f e to leave the l i v e r to any s i g n i f i c a n t extent. Since N-hydroxyacetaminophen has a r e l a t i v e l y long h a l f - l i f e i n v i t r o , the p o s s i b i l i t y that the h e p a t o t o x i c i t y of acetaminophen might be mediated mainly through t h i s metabolite became q u e s t i o n a b l e . Recent s t u d i e s have shown that hamster l i v e r microsomes convert N-hydroxyphenacetin but not acetaminophen to N-hydroxyacetaminophen even though c o n s i d e r b l y more acetaminophen i s c o v a l e n t l y bound to microsomal p r o t e i n s than i s N-hydroxyphenacetin (35). Moreover, the chemically r e a c t i v e metabolite of acetaminophen i s apparently not formed by way of acetaminophen epoxide because the formation of 3-hydroxyacetaminophen i s not blocked by g l u t a t h i o n e , a s c o r b i c a c i d or epoxide hydrolase and covalent binding of acetaminophen i s not blocked by superoxide dismutase (36)· Thus, the c h e m i c a l l y r e a c t i v e metabolite of acetaminophen remains u n i d e n t i f i e d . It i s s t i l l p o s s i b l e that the intermediate i s N-acetylimidoquinone (N-acetyl-p-benzoquinoneimine) because i t reacts with g l u t a thione to form a glutathione-acetaminophen conjugate, and i s r e a d i l y reduced to acetaminophen by a s c o r b i c a c i d . I f Nacetylimidoquinone i s the major r e a c t i v e m e t a b o l i t e , however, i t must be formed by a h i t h e r t o unknown mechanism. These s t u d i e s thus i n d i c a t e d that the l i v e r n e c r o s i s caused by acetaminophen i n mice i s mediated by a chemically r e a c t i v e metabolite that combines with g l u t a t h i o n e conjugate to form a conjugate, which u l t i m a t e l y i s excreted as a mercapturic acid. The s t u d i e s f u r t h e r i l l u s t r a t e d how a change i n the a c t i v i t y of an enzyme that c a t a l y z e s the formation of a minor t o x i c metabolite can markedly a f f e c t the t o x i c i t y without s i g n i f i c a n t l y a f f e c t i n g the b i o l o g i c a l h a l f - l i f e of the parent drug. The f i n d i n g that g l u t a t h i o n e i s markedly decreased before the covalent binding of the a c t i v e metabol i t e of acetaminophen to p r o t e i n becomes a p p r e c i a b l e l e d to the concept of a "dose t h r e s h o l d " f o r the t o x i c i t y . In mice the "dose t h r e s h o l d " i s r e l a t e d to the f r a c t i o n of the dose that i s converted to the r e a c t i v e metabolite (Ratio A) and the amount of g l u t a t h i o n e i n i t i a l l y present i n the l i v e r . But i t should be pointed out that the reason f o r "dose t h r e h s o l d s " may d i f f e r i n other animal s p e c i e s . As the dose i s increased, the f r a c t i o n of the dose i s converted to acetaminophen s u l f a t e decreases, i n d i c a t i n g that t h i s pathway of i n a c t i v a t i o n becomes saturated e i t h e r because the c o n c e n t r a t i o n of acetaminophen i n l i v e r exceeds the KJJJ of the s u l f o t r a n s f e r a s e or because the s y n t h e s i s of

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l

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231

,

3 -phosphoadenosine-5 -phosphosulfate (PAPS), the cosubsubstrate of the enzyme, becomes r a t e - l i m i t i n g . As the dose i s increased f u r t h e r the c o n c e n t r a t i o n of acetaminophen may exceed K of g l u c u r o n y l t r a n s f e r a s e i n l i v e r of some animal species. Indeed, the s a t u r a t i o n of both of these enzyme systems may account i n part f o r the f i n d i n g that the apparent h a l f - l i f e of acetaminophen (10) and the f r a c t i o n of the dose excreted as the mercapturic a c i d (37^,38) increases as the dose i s increased i n man. The f i n d i n g that c y s t e i n e can prevent the l i v e r n e c r o s i s caused by acetaminophen i n mice (17) l e d to the p o s s i b i l i t y that t h i o compounds might be u s e f u l as a n t i d o t e s , provided that they are administered while the acetaminophen i s being metabolized. Unfortunately, c y s t e i n e i s a r a t h e r i n e f f e c t i v e a n t i d o t e except when i t i s administered i n t r a p e r i t o n e a l l y because i t i s incorporated i n t o p r o t e i n by a l l t i s s u e s of the body and thus i s subject to a k i n d of f i r s t pass e f f e c t by these t i s s u e s . Most of the emphasis, t h e r e f o r e , has been toward the development of a n t i d o t e s that serve as precursors of c y s t e i n e (such as methionine and N - a c e t y l c y s t e i n e ) and thus of g l u t a t h i o n e or as a l t e r n a t i v e n u c l e o p h i l e s that combine with the chemically r e a c t i v e metabolite. m

Cysteamine apparently i s an e f f e c t i v e a n t i d o t e not only i n mice (39) but a l s o i n man (40)· At f i r s t i t was assumed that t h i s compound exerted i t s e f f e c t by s e r v i n g as an a l t e r n a t i v e n u c e l o p h i l e i n the i n a c t i v a t i o n of the chemically r e a c t i v e metabolite. I t i s a l s o p o s s i b l e , however, that cysteamine may act by i n h i b i t i n g the formation of the chemi c a l l y r e a c t i v e metabolite (41) and by s e r v i n g as a precursor of s u l f a t e , r e q u i r e d f o r t h e ~ o r m a t i o n of PAPS. Unfortunately, i t i s d i f f i c u l t to d i f f e r e n t i a t e among these mechanisms. The evidence c i t e d i n support of the concept that cysteamine i n h i b i t s the formation of the r e a c t i v e metabolite i s based p r i m a r i l y on the f i n d i n g that cysteamine decreases the exc r e t i o n of the g l u t a t h i o n e conjugate i n t o b i l e and of the c y s t e i n y l conjugate and mercapturic a c i d i n t o u r i n e . Moreover, no evidence was obtained i n d i c a t i n g that a cysteamine conjugate of acetaminophen i s excreted i n t o b i l e or u r i n e . However, these r e s u l t s are not d e f i n i t i v e . Cysteamine would cause a decrease i n the e x c r e t i o n of the g l u t a t h i o n e i n t o b i l e and c y s t e i n e conjugates and the mercapturic a c i d i n t o u r i n e even i f i t were to exert i t s p r o t e c t i v e e f f e c t s o l e l y by combining with the chemically r e a c t i v e m e t a b o l i t e . Moreover, i t i s questionable whether the c y s t e i n e conjugate of acetaminophen would be r a p i d l y excreted i n t o b i l e or u r i n e before i t i s converted to other substances by enzymes such as monoamine oxidase. Furthermore, the f a c t that high concentrations of cysteamine i n h i b i t the h y d r o x y l a t i o n of a c e t a n i l i d e i n v i t r o (41) may or may not be r e l e v a n t because i t i s not known whether the formation of the chemically r e a c t i v e metabolite of acetaminophen i s c a t a l y z e d by the

232


same enzyme that hydroxylates a n i l i d e or whether the conc e n t r a t i o n s of cysteamine achieved i n v i v o approach those used i n v i t r o * I t i s a l s o questionable that the increase i n s u l f a t e derived from cysteamine would a f f e c t R a t i o A by more than a few percent. I t seems to me that the mechanism by which cysteamine exerts i t s p r o t e c t i v e e f f e c t must remain open. N-Acetylcysteine a l s o prevents the l i v e r n e c r o s i s caused by acetaminophen i n animals (42,43,440 and man (45,46)· But again, the mechanism i s not e n t i r e l y c l e a r . It i s possible that N - a c e t y l c y s t e i n e may combine d i r e c t l y with the chemically r e a c t i v e metabolite to form the mercapturic a c i d . I t i s a l s o p o s s i b l e , however, that N - a c e t y l c y s t e i n e i s deacetylated to c y s t e i n e and then converted to g l u t a t h i o n e (47) or o x i d i z e d to s u l f a t e (48). A l l of these mechanisms would tend to decrease the t o x i c i t y of acetaminophen. Thus our attempts to i d e n t i f y the t o x i c chemically r e a c t i v e metabolite of acetaminophen have been e l u s i v e . But imagine the greater d i f f i c u l t y i n e l u c i d a t i n g t o x i c metabolites when the substance can be converted to s e v e r a l d i f f e r e n t chemically r e a c t i v e metabolites or to the same chemically r e a c t i v e metabol i t e by d i f f e r e n t mechanisms. Phenacetin can be converted to chemically r e a c t i v e metabol i t e s that combine with g l u t a t h i o n e through at l e a s t four d i f f e r e n t pathways ( F i g . 3 ) . 1) Phenacetin i s converted to acetaminophen (9) which i s subsequently a c t i v a t e d to a chemi c a l l y r e a c t i v e metabolite that combines with g l u t a t h i o n e (21). In t h i s pathway the phenolic oxygen i n the acetaminophen-SG conjugate o r i g i n a t e s from the ethoxy oxygen of phen a c e t i n (22^,24^). 2) Phenacetin i s converted to an intermediate we b e l i e v e to be phenacetin-3,4-epoxide. The intermediate decomposes to another chemically r e a c t i v e metabolite that r e a c t s with g l u t a t h i o n e to form an acetaminophen-SG conjugate. E x a c t l y 50% of the phenolic oxygen i n the conjugate formed by t h i s pathway o r i g i n a t e s from atmospheric oxygen and the other 50% o r i g i n a t e s from phenacetin (22,24)· 3) Phenacetin i s converted to N-hydroxyphenacetin (38). In turn the Nhydroxyphenacetin can be transformed to N - s u l f a t e and N0glucuronide conjugates which decompose to a chemically r e a c t i v e metabolite that r e a c t s with g l u t a t h i o n e to form an acetaminophen-SG conjugate (50)· The phenolic oxygen i n the conjugate formed by t h i s pathway o r i g i n a t e s from water (24). 4) Phenacetin may be converted to N-hydroxyphenacetin as i n pathway 3 but then undergoes o x i d a t i v e d e a l k y l a t i o n to a chemically r e a c t i v e metabolite that reacts with g l u t a t h i o n e to form an acetaminophen-SG conjugate (32,35). The phenolic oxygen i n the conjugate formed by t h i s pathway presumably o r i g i n a t e s from phenacetin. Another pathway f o r the formation of a chemically r e a c t i v e metabolite may be p o s t u l a t e d . In t h i s pathway acetaminophen i s converted to 3-hydroxyacetamino-

14.

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Figure 3.

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Pathways of phenactin metabolism leading to the formation of glutatione conjugates

233

234


phen (36,51), which i s a c a t e c h o l and thus may be o x i d i z e d to a quinone by superoxide (52,53). Although my Laboratory has used these p r i n c i p l e s to study the t o x i c i t i e s caused by l a r g e doses of drugs, there i s every reason to b e l i e v e that these p r i n c i p l e s w i l l be e q u a l l y a p p l i c a b l e i n studying species d i f f e r e n c e s i n the e f f e c t s of p e s t i c i d e s . Indeed, i t i s now b e l i e v e d that compounds such as p i p e r o n y l butoxide and parathion i n h i b i t cytochrome P-450 enzymes through the formation of c h e m i c a l l y r e a c t i v e metabolites. The s p e c i f i c i t y of the e f f e c t s of these sub stances presumably occurs e i t h e r because the chemically r e a c t i v e metabolites have an unusually high a f f i n i t y f o r the cytochrome P-450 enzymes or because they are so s h o r t l i v e d that they never leave the immediate environment of the a c t i v e s i t e s of the enzymes. The use of other " s u i c i d e enzyme i n h i b i t o r s " o f f e r s e x c i t i n g p o s s i b i l i t i e s .

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

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