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1 Conjugation Mechanisms of Xenobiotic Metabolism: Mammalian Aspects John Caldwell

Downloaded by MCGILL UNIV on December 9, 2012 | http://pubs.acs.org Publication Date: January 24, 1986 | doi: 10.1021/bk-1986-0299.ch001

Department of Pharmacology, St. Mary's Hospital Medical School, University of London, London W2 1PG, England

Living organisms of all types exist in a chemical environment, made up of nutrients (macronutrients, micronutrients and trace elements) and anutrients or xenobiotics. These latter comprise those compounds from which organisms are (virtually) unable to utilize for energy-yielding metabolism, and may be broadly divided into two classes, natural and synthetic. Living organisms may be exposed to xenobiotics (a) deliberately, due to their use as drugs, pesticides, herbicides etc., (b) coincidentally in the diet, most notably in the consumption of plant foods by animals of a l l types, or (c) accidentally, from industrial, agricultural or other sources. It is the purpose of this review to consider the fates of such xenobiotics in living organisms, as an introduction to the topic of this Symposium volume, concentrating in particular upon the metabolic conjugation reactions as they occur in mammals. When a xenobiotic enters a living organism, i t may undergo one or more of a number of fates, as follows : 1.

Enzymic metabolism leading to elimination

2.

Enzymic metabolism leading to accumulation

3.

Elimination unchanged

4.

Spontaneous (non-enzymic) chemical transformation

5.

Accumulation unchanged

Although the last two options are very important when they occur, as exemplified by the unfortunate cases of thalidomide (1) and the polyhalogenated biphenyls (2) respectively, the great majority of xenobiotics undergo enzymic metabolism and/or are eliminated unchanged. In animals, in most cases, metabolism favours the elimination of the compound by enhancing its polarity 0097-6156/86/0299-0002$07.50/0 © 1986 American Chemical Society

In Xenobiotic Conjugation Chemistry; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

1. CALDWELL

Xenobiotic Metabolism: Mammalian Aspects


and water s o l u b i l i t y . Only r a r e l y does m e t a b o l i s m cause the retention of a x e n o b i o t i c w i t h i n an a n i m a l : t h i s may o c c u r in one of two ways (a) the f o r m a t i o n of lipophilic metabolites (3) or (b) m e t a b o l i s m to r e a c t i v e s p e c i e s , which b i n d c o v a l e n t l y t o t a r g e t s i t e s w i t h i n body c o n s t i t u e n t s ( 4 ) . Only i n v e r y r a r e cases are s u c h r e a c t i o n s of q u a n t i t a t i v e importance in the d i s p o s i t i o n of a x e n o b i o t i c , a l t h o u g h they a r e of c o u r s e o f t e n of b i o l o g i c a l s i g n i f i c a n c e , notably i n t o x i c i t y (5). The s i t u a t i o n in plants obviously differs from the above* Lacking the excretion mechanisms of a n i m a l s , m e t a b o l i s e d xenobiotics are r e t a i n e d by the p l a n t , f r e q u e n t l y s e q u e s t e r e d i n l i g n i n and o t h e r cell constituents which p r e v e n t any interference with cell f u n c t i o n (6). Any s u r v e y , however c u r s o r y , of the now s u b s t a n t i a l l i t e r a t u r e on the metabolic f a t e of x e n o b i o t i c s w i l l s e r v e t o i l l u s t r a t e the p l e t h o r a of p o s s i b l e r e a c t i o n s . One of the major contributions of R. T. Williams was t o d i s c e r n t h a t t h e s e r e a c t i o n s may be classified i n t o two distinct types which g e n e r a l l y occur s e q u e n t i a l l y (7). The x e n o b i o t i c i n i t i a l l y undergoes a Phase I, or functlonalization, r e a c t i o n of oxidation, reduction or hydrolysis the p r o d u c t of w h i c h i s s u b j e c t e d t o a Phase II, or conjugation, reaction* These l a t t e r a r e b i o s y n t h e s e s , i n w h i c h the xenobiotic ( o r exocon) i s l i n k e d w i t h an endogenous conjugating agent ( o r endocon) to g i v e a c h a r a c t e r i s t i c p r o d u c t termed a c o n j u g a t e * In some c a s e s , t h i s b l p h a s i c sequence does not o p e r a t e : some Phase I m e t a b o l i t e s may be e l i m i n a t e d w i t h o u t being c o n j u g a t e d , w h i l e o t h e r x e n o b i o t i c s may undergo Phase II metabolism d i r e c t l y * The e l i m i n a t i o n p r o d u c t s of x e n o b i o t i c s may thus comprise the unchanged compound, Phase I m e t a b o l i t e s , Phase I I m e t a b o l i t e s and the p r o d u c t s of the b l p h a s i c sequence* It is most commonly the c a s e t h a t x e n o b i o t i c s a r e e l i m i n a t e d in the form of c o n j u g a t e s * Compounds u n d e r g o i n g m e t a b o l i c c o n j u g a t i o n must p o s s e s s within their s t r u c t u r e s a f u n c t i o n a l group a p p r o p r i a t e f o r combination w i t h the c o n j u g a t i n g agent ( 8 ) . T h i s i s g e n e r a l l y i n t r o d u c e d by Phase I m e t a b o l i s m , and may e i t h e r be c h e m i c a l l y stable e.g. phenolic, a l c o h o l i c or c a r b o x y l i c h y d r o x y l , amine, t h i o l or a c y c l i c heteroatom, or c h e m i c a l l y r e a c t i v e e.g. arene oxide, or carbonium i o n . It i s now g e n e r a l l y a c c e p t e d t h a t t h e r e o c c u r i n mammals s i x major conjugation reactions a v a i l a b l e to xenobiotics (9). In each case, the s u b s t r a t e and c o - f a c t o r requirements, enzymic basis and phylogenetic distribution are (reasonably) well u n d e r s t o o d . These s i x r e a c t i o n s e a c h have a w e l l - d e f i n e d r o l e i n the m e t a b o l i s m of endogenous compounds and i n b i o s y n t h e s e s . All i n v o l v e the p a r t i c i p a t i o n of a t r a n s f e r a s e enzyme, w h i c h e x h i b i t s high s p e c i f i c i t y f o r the c o n j u g a t i n g agent i n question. Each reaction i s a biosynthesis, and i s e n e r g y - r e q u i r i n g : t h i s is provided e i t h e r by the c o n j u g a t i n g agent b e i n g p r e s e n t in an a c t i v a t e d form (most f r e q u e n t l y as a n u c l e o t i d e ) or by the prior a c t i v a t i o n of the x e n o b i o t i c s u b s t r a t e . The major r e a c t i o n s a r e listed i n T a b l e I , w h i c h g i v e s d e t a i l s of t h e i r energy sources and endogenous r o l e ( s ) .


3

XENOBIOTIC CONJUGATION CHEMISTRY


4

In a d d i t i o n t o the ' c l a s s i c a l ' c o n j u g a t i o n r e a c t i o n s l i s t e d in Table 1, there o c c u r s a l s o an I n c r e a s i n g number of s o - c a l l e d ' n o v e l ' c o n j u g a t i o n s (10, 11). These a r e added t o r e g u l a r l y , as a consequence of the e v e r more r i g o r o u s i n v e s t i g a t i o n of both excreted metabolites and of t i s s u e r e s i d u e s of xenobiotics by increasingly sophisticated analytical methodology. The novel r e a c t i o n s a r e those p r e s e n t l y viewed as b e i n g r e s t r i c t e d i n t h e i r occurrence to p a r t i c u l a r combinations of substrate(s) and species. This status i s due s i m p l y t o the current lack of knowledge (10), and some, a t l e a s t , of these r e a c t i o n s may be expected to a t t a i n the status of major r e a c t i o n s as more information i s acquired. Indeed, i t a l r e a d y seems t h a t the f o r m a t i o n of m e t h y l t h i o c o n j u g a t e s , a r e a c t i o n f i r s t observed i n the mid-1970s (12), may be a g e n e r a l r e a c t i o n of xenobiotic metabolism (13). Recent developments i n the d i s c o v e r y of n o v e l reactions, and i n the a n a l y t i c a l methodology upon w h i c h they a r e based, a r e covered i n d e t a i l elsewhere i n t h i s volume, and thus w i l l not be d e a l t w i t h f u r t h e r h e r e . Historical

perspective

B e f o r e p r o c e e d i n g t o a r e v i e w of the major c o n j u g a t i o n reactions extant i n mammals, i t i s of some I n t e r e s t to consider the h i s t o r i c a l e v o l u t i o n of our knowledge of t h i s important group of pathways. The c o n j u g a t i o n r e a c t i o n s were the f i r s t r e a c t i o n s of foreign compound m e t a b o l i s m t o be d i s c o v e r e d , i f only f o r the s i m p l e r e a s o n t h a t they produce the f i n a l e l i m i n a t i o n p r o d u c t s of xenobiotics. However, the e a r l y h i s t o r y of t h e i r s t u d y d e r i v e s from a time l o n g b e f o r e the d e l i n e a t i o n of our present subdiscipline of 'drug metabolism', when p i o n e e r i n g workers dealt with the a p p l i c a t i o n of c h e m i c a l principles to p h y s i o l o g i c a l problems. The study of f o r e i g n compound m e t a b o l i s m led to numerous important d i s c o v e r i e s i n b i o c h e m i s t r y (14, 15), beside which their significance, viewed simply i n terms of 'drug metabolism', pales i n comparison. T h i s i s e x e m p l i f i e d by the sequence of e v e n t s l e a d i n g t o the d i s c o v e r y of the c o n j u g a t i o n of b e n z o i c a c i d w i t h g l y c i n e , the h i p p u r i c a c i d s y n t h e s i s , which was not m e r e l y the f i r s t r e a c t i o n of d r u g metabolism, but w h i c h was the first b i o s y n t h e s i s of any k i n d t o be d i s c o v e r e d and which could not be reproduced i n the test tube f o r some years afterwards. The g r e a t Swedish chemist C. W. Scheele d i s c o v e r e d benzoic a c i d in gum benzoin i n 1775, by s u b l i m a t i o n of the resin (16). Applying t h i s t e c h n i q u e t o many n a t u r a l m a t e r i a l s , between 1770 and 1786, he a l s o d i s c o v e r e d t a r t a r i c , lactic, oxalic, c i t r i c , gallic and m a l i c a c i d s , and i n 1776 he found b e n z o i c acid i n human u r i n e ( 1 6 ) . F o l l o w i n g t h i s i n 1784, R o u e l l e (17) found benzoic a c i d i n the u r i n e of cows, but i n 1799, Fourcroy and V a u q u e l i n (18) showed t h a t the a c i d o b t a i n e d by these workers was not b e n z o i c a c i d , but a n o t h e r , s i m i l a r a c i d , which they c o u l d not identify.


1. CALDWELL



The founder of modern o r g a n i c c h e m i s t r y and pioneer of the application of chemical p r i n c i p l e s to p h y s i o l o g i c a l problems, Justus von L l e b l g , became i n t e r e s t e d i n t h e s e a c c o u n t s and in 1830 he r e p e a t e d the experiments of F o u r c r o y and V a u q u e l i n (19)* He found a new nitrogen-containing acid i n horse u r i n e , a compound of b e n z o i c a c i d w i t h an unknown n i t r o g e n o u s body, which he termed ' h i p p u r i c a c i d ' from the Greek f o r ' h o r s e ' and ' u r i n e ' . In t h i s paper, L l e b l g s p e c u l a t e d on the o r i g i n of h i p p u r i c a c i d , and n o t e d t h a t b e n z o i c a c i d as s u c h was not p r e s e n t i n the diet of h o r s e s . The honour of the d i s c o v e r y of s y n t h e s i s of h i p p u r i c acid from i n g e s t e d b e n z o i c a c i d s h o u l d be a c c r e d i t e d t o the B r i t i s h s u r g e o n Alexander Ure, at t h a t time on the s t a f f of Guy's H o s p i t a l in London. Ure attempted to t r e a t gout w i t h benzoic a c i d and following the o r a l a d m i n i s t r a t i o n of one s c r u p l e (1.3g) he Isolated 15 g r a i n s (« 0.97g) of h i p p u r i c a c i d f r o m the urine. The p u b l i c a t i o n of Ure's f i n d i n g i n the London M e d i c a l G a z e t t e i n 1841 (20) s t i m u l a t e d f u r t h e r work i n Germany and L i e b i g ' s close c o l l a b o r a t o r F r i e d r i c h Wohler prompted h i s s t u d e n t W i l h e l m K e l l e r (21) to repeat the investigations of Ure, by selfexperimentatlon. I t was t h i s s t u d y w h i c h s e r v e d t o prove that h i p p u r i c a c i d i s i n d e e d formed from b e n z o i c a c i d . The exact nature of h i p p u r i c a c i d was d i s c e r n e d i n 1845 by D e s s a i g n e s (22) who showed t h a t the n i t r o g e n o u s body l i n k e d with b e n z o i c a c i d was g l y c i n e . The e x i s t e n c e of g l y c i n e had been known s i n c e 1820, when Braconnot found i t i n g e l a t i n e (23). However, the c h e m i c a l s y n t h e s i s of h i p p u r i c a c i d from benzoic acid and g l y c i n e was not a c h i e v e d u n t i l 1857, when Dessaignes (24) fused the constituents i n a sealed tube reaction in a furnace. Although the work of Ure and K e l l e r had shown t h a t the administration of b e n z o i c a c i d l e d t o the e x c r e t i o n of hippuric acid, the n a t u r a l o c c u r r e n c e of the l a t t e r i n the u r i n e c o u l d be considered to render t h e i r s t u d i e s e q u i v o c a l . The unambiguous proof of the l i n k a g e of an exogenous c a r b o x y l i e a c i d could be l i n k e d w i t h g l y c i n e i n t h e body was p r o v i d e d by Cesar B e r t a g n i n l , who showed t h a t ^ . - a i t r o b e n z o i c and o - h y d r o x y b e n z o i c (salicylic) a c i d s (25, 26). were b o t h c o n v e r t e d i n p a r t t o the c o r r e s p o n d i n g hippuric acids. In these s t u d i e s , the n i t r o and phenolic hydroxy1 groups f u n c t i o n e d as c h e m i c a l l a b e l s , much as we would use i s o t o p e s t o d a y . Following this, from s t u d i e s of normal and pathological body fluids, and f r o m e x a m i n a t i o n of the m e t a b o l i c f a t e s of various organic compounds, between 1870 and 1900 the remainder of the major c o n j u g a t i o n r e a c t i o n s were d i s c o v e r e d . These a r e l i s t e d i n T a b l e I I . Between 1900 and 1970, new d i s c o v e r i e s o c c u r r e d very infrequently, but i n the l a s t 15 y e a r s the a p p l i c a t i o n of modern analytical techniques (see above) has added new examples at a steady r a t e . The more r e c e n t h i s t o r y of the c o n j u g a t i o n r e a c t i o n s r e v e a l s t h a t their s t u d y has f o l l o w e d a sequence i n which the y e a r s f o l l o w i n g


5


Table I. C l a s s i f i c a t i o n of the Major Conjugation Reactions Reaction

High-energy

intermediate


Reactions with activated conjugating

Endogenous roles

agents

Glucuronidation

UDP glucuronic a c i d

Biosynthesis, detoxicatlon

Sulfation

PAPS


Methylation

S-Adenosyl methionine


Acetylation

Acetyl CoA

Biosynthesis, Intermediary metabolism

Reactions with activated xenobiotic

Glutathione conjugation

Epoxides, nitrenlum ions e t c .

Maintenance of redox potent i a l , leukotriene synthesis

Amino a c i d conjugation

Xenobiotic a c y l CoAs

Biosynthesis, detoxicatlon (esp. i n amino a c i d u r i a s )

Table I I .

The D i s c o v e r y

o f the Major C o n j u g a t i o n s

Conjugation

Author and date

Glycine

Keller

Sulfate

Baumann (1876a)

Glucuronic

acid

Ornithine Mercapturic

acid

(1842)

Jaffe

(1874)

Jaffe

(1877)

J a f f e (1879); Baumann & P r e u s s e (1879)

Methylation

His

Acetylation

Cohn (1893)

Glutamine

T h i e r f e l d e r and Sherwin (1914)

Taurine

James (1971)

(1887)

Based on r e f . 27


CALDWELL



t h e i r i n i t i a l d i s c o v e r y have been devoted t o the c o n s o l i d a t i o n of knowledge c o n c e r n i n g t h e i r o c c u r r e n c e ( 2 7 ) , i n v o l v i n g the s e a r c h for new x e n o b i o t i c s u b s t r a t e s and the examination of v a r i o u s animal s p e c i e s f o r the o c c u r r e n c e of the r e a c t i o n i n q u e s t i o n * Following this, the p o s t - W o r l d War 2 years have seen the development of knowledge c o n c e r n i n g the b i o c h e m i s t r y of these r e a c t i o n s (28, 29), both from the v i e w p o i n t s of the enzymology of the t r a n s f e r a s e s and of the s u p p l y and regulation of the conjugating agent* Some of these discoveries have had fundamental importance o u t s i d e drug metabolism a l o n e , n o t a b l y the discovery of Coenzyme A by Lipmann (30) a r i s i n g from work on the a c e t y l a t i o n of s u l f o n a m i d e s . Two c h a r a c t e r i s t i c f e a t u r e s mark c u r r e n t a c t i v i t y i n the s t u d y of the conjugations (a) the a p p l i c a t i o n of the techniques of molecular b i o l o g y and (b) an enhanced a p p r e c i a t i o n of their pharmacological and t o x i c consequences. I t i s t o be expected that the next few y e a r s w i l l see many developments a r i s i n g from the former i n n o v a t i o n ( 3 1 ) , but i t i s the l a t t e r a r e a t h a t most p r o g r e s s has been made i n r e c e n t y e a r s . From the 1870s onwards, v a r i o u s e a r l y workers concerned themselves w i t h the consequences of x e n o b i o t i c m e t a b o l i s m , i n particular n o t i n g t h a t c o n j u g a t e s were markedly l e s s t o x i c than their parent f o r e i g n compounds ( 2 7 ) . T h i s l e d t o the i d e a t h a t this process r e s u l t e d i n ' d e t o x i c a t l o n ' (a t r a n s l a t i o n of the German ' e n t g i f t u n g ' ) , and t h i s became v e r y w i d e l y a c c e p t e d . The concept of d e t o x i c a t l o n reached its most sophisticated development i n the c h e m i c a l defence h y p o t h e s i s of CP. Sherwin (27). Since the 1920s, i t has been i n c r e a s i n g l y r e a l i z e d t h a t the Phase I reactions are o f t e n i n v o l v e d i n i n c r e a s i n g the activity o f x e n o b i o t i c s , and thus the i d e a of t h e s e r e a c t i o n s being d e t o x l c a t i o n s was abandoned. However, t h i s concept was much l o n g e r l a s t i n g i n the case of the c o n j u g a t i o n s : t h e s e g i v e r i s e to s t a b l e , r e a d i l y e x c r e t e d , i n a c t i v e m e t a b o l i t e s and i t was not u n t i l the l a t e 1970s t h a t t h i s view came t o be m o d i f i e d . It was realized t h a t due to i n t e r - s p e c i e s differences i n the o c c u r r e n c e of t h e s e r e a c t i o n s or the l i m i t e d c a p a c i t y of certain conjugations, the d e t o x i c a t l o n f u n c t i o n of the conjugations failed more f r e q u e n t l y than had been a p p r e c i a t e d h i t h e r t o . In addition, a number of i n s t a n c e s of conjugates being active m e t a b o l i t e s had accumulated i n the l i t e r a t u r e , and the c o l l a t i o n of i n f o r m a t i o n on these two a s p e c t s s e r v e d t o f o c u s the a t t e n t i o n of pharmacologists and t o x i c o l o g i s t s on c o n j u g a t i v e metabolism The l a s t f i v e y e a r s have seen a g r e a t i n c r e a s e i n work i n this a r e a , w h i c h c o n t i n u e s t o grow.

The major c o n j u g a t i o n r e a c t i o n s It i s the purpose of the remainder of t h i s r e v i e w t o s u r v e y the major conjugation reactions, i n terms of their substrate v e r s a t i l i t y , enzymic mechanism and d i s t r i b u t i o n amongst mammalian



8 species, and consequences•


Glucuronic acid

to

comment

briefly

upon

their

biological

conjugation

The g l u c u r o n i c a c i d c o n j u g a t i o n i s the most v e r s a t i l e of the conjugation r e a c t i o n s i n terms of the range of xenobiotic substrates i t may a c c e p t and i t s w i d e s p r e a d d i s t r i b u t i o n t h r o u g h species and t i s s u e s . The g l u c u r o n i c a c i d r e s i d u e Incorporated into the conjugate derives from the nucleotide uridine diphosphate g l u c u r o n i c a c i d (UDPGA) and i s t r a n s f e r r e d t o the x e n o b i o t i c under the I n f l u e n c e of the UDP g l u c u r o n y l t r a n s f e r a s e s (UDPGTs) ( 3 2 ) . The g l u c u r o n i c a c i d r e s i d u e may be a t t a c h e d to one of a wide range of f u n c t i o n a l groups ( 3 3 ) , w h i c h a r e listed i n T a b l e I I I . A l t h o u g h the main c l a s s e s of g l u c u r o n i d e have been known f o r more than a c e n t u r y , i t i s of i n t e r e s t t o note t h a t new classes of glucuronide are s t i l l being discovered e.g. the q u a t e r n a r y amino g l u c u r o n i d e s of drugs s u c h as c y p r o h e p t l d i n e and the C - g l u c u r o n l d e s of the p y r a z o l o n e s . I n UDPGA, the pyranose r i n g of g l u c u r o n i c a c i d i s i n the alpha form i . e . the p r o t o n s on C - l and C-2 a r e e l s to e a c h other. During the enzymic t r a n s f e r t o the acceptor substrate, the anomerlc c e n t r e undergoes i n v e r s i o n such t h a t , i n the c o n j u g a t e , glucuronic acid i s of the b e t a - c o n f i g u r a t i o n . The xenobiotic r e s i d u e i s always l i n k e d t o the h y d r o x y l group on C-l, although in the case o f the e s t e r g l u c u r o n i d e s t h e r e may o c c u r subsequent a c y l group m i g r a t i o n to the h y d r o x y l groups on C-2, C-3 and C-4. Any such m i g r a t i o n occurs after formation of the 1-0-acyl glucopyranosiduronates, and other Isomers a r e not formed enzymatically (34). UDPGT i s a membrane-bound enzyme, found particularly in the e n d o p l a s m i c r e t i c u l u m of the l i v e r and numerous o t h e r t i s s u e s ( 3 2 ) . The a c t i v i t y of the enzyme i s a t l e a s t p a r t l y l a t e n t ( 3 5 ) , being enhanced by a v a r i e t y of agents a b l e t o d i s r u p t membrane s t r u c t u r e ( l i p o l y t i c enzymes, d e t e r g e n t s , o r g a n i c s o l v e n t s e t c . ) . UDP-N-acetylglucosamine p l a y s a r o l e i n d e t e r m i n i n g the latency of the enzyme i n v i v o . E v i d e n c e showing t h a t UDPGT e x h i b i t s a t least f u n c t i o n a l h e t e r o g e n e i t y has been accumulated over many years, and I n c l u d e s f i n d i n g s of s p e c i e s and s t r a i n d i f f e r e n c e s , substrate s p e c i f i c i t y , d i f f e r e n t i a l ontogenesis, induction and i n h i b i t i o n , t i s s u e d i s t r i b u t i o n and i n v i v o a c t i v a t i o n ( 3 2 ) . The molecular b a s i s of t h i s h e t e r o g e n e i t y has been hard t o e s t a b l i s h owing t o the d i f f i c u l t i e s of working w i t h membrane-bound enzymes. At the p r e s e n t time, t h r e e c l e a r l y - d e f i n e d , s e p a r a t e forms have been purified to apparent heterogeneity, these being differentially inducible and having different, albeit overlapping, substrate specificities (31). The e x i s t e n c e of further forms has been suggested by s t u d i e s of the i n d u c t i o n of UDPGT and of i t s s u b s t r a t e s p e c i f i c i t y . The occurrence of m u l t i p l e UDPGTs w i t h overlapping substrate specificities makes assays of glucuronidation a c t i v i t y of particular substrates by subcellular fractions u n r e l i a b l e as


I. CALDWELL


T a b l e I I I . Types o f Compounds G i v i n g R i s e to G l u c u r o n i c A c i d Conjugates

Functional

group

Example


Hydroxyl Primary a l c o h o l Secondary a l c o h o l Tertiary alcohol A l i c y c l i c alcohol Terpenoid a l c o h o l Phenol T e r p e n o i d phenol Enol A l p h a t i c hydroxylamine A r o m a t i c hydroxylamine Hydroxamic a c i d Carboxylic

acid

Alkyl Aromatic Heterocyclic Arylacetic Arylpropionic Aryloxybutyric Carbamic a c i d Amino

trichloroethanol propranolol tert-butanol cyclohexanol menthol phenol eugenol 4-hydroxycoumarin ^-hydroxychlorphentermine 2-naphthylhydroxylamine Jjf-hy droxy-2-acetamidof l u o r e n e

2-ethylhexanoic a c i d benzoic a c i d nicotinic acid Indole-3-acetic acid hydratropic acid clofibric acid t o c a i n i d e carbamate

functions

Aromatic Azaheterocycle Carbamate Sulphonamide Hydroxy lamine j j Tertiary aliphatic Urea Sulphur

aniline sulphisoxazole meprobamate

8ulphadimethoxine 2J-hy droxy-2-acetamidof l u o r e n e cyproheptadine dulcin

functions

Thiol Dithioic acid

2-mercaptobenzothiazole N,N-diethyldithiocarbamic acid

Carbon c e n t r e s Pyrazolone r i n g

p h e n y l b u t a z one

Adapted

from r e f . _33


9

H)


indicators of the presence of particular isozymes. This s i t u a t i o n w i l l remain u n t i l immunochemical or m o l e c u l a r biology techniques permit the a s s a y of i n d i v i d u a l isozymes, or until i s o z y m i c - s p e c i f i c s u b s t r a t e s a r e d i s c o v e r e d . To reduce c o n f u s i o n i n the i n t e r i m , s t a n d a r d i z e d a s s a y c o n d i t i o n s have r e c e n t l y been recommended ( 3 6 ) .


Glucuronic acid conjugation i s widely d i s t r i b u t e d through the animal kingdom. I t i s i n v o l v e d i n the m e t a b o l i s m of f o r e i g n compounds i n mammals, b i r d s , f i s h , amphibians and r e p t i l e s , but not i n i n s e c t s or m o l l u s e s ( 3 3 ) . I n these l a t t e r i n s t a n c e s , i t is replaced by the analogous r e a c t i o n of glucose conjugation (33). G l u c u r o n i d a t l o n has a l s o been r e p o r t e d t o o c c u r i n p l a n t s (33). Virtually a l l mammals a r e able to form glucuronic acid conjugates, but t h e r e do e x i s t c e r t a i n s p e c i e s , and g e n e t i c a l l y stable mutant8 w i t h i n s p e c i e s , which a r e marked by an ( a t l e a s t p a r t i a l ) i n a b i l i t y to e f f e c t t h i s r e a c t i o n ( 3 3 ) . These include t h r e e examples o f s u b s t r a t e s whose g l u c u r o n i d a t l o n o n l y o c c u r s i n primate species (a) cyproheptadine quaternary N-glucuronidation (b) C-glucuronidation of phenylbutazone and other pyrazolones (both only found i n human and chimpanzee) and ( c ) the N glucuronidatlon of s u l f a d l m e t h o x i n e and some o t h e r sulfonamides (found i n a l l p r i m a t e s ) . Probably the best example of a 'species defect' i n drug m e t a b o l i s m i s the inability of the domestic cat to form glucuronides of many compounds which a r e e x t e n s i v e l y m e t a b o l i s e d along t h i s pathway i n most o t h e r s p e c i e s . T h i s was originally noted by Robinson and W i l l i a m s (37) i n 1956, and many subsequent s t u d i e s have c o n f i r m e d and extended t h e i r o b s e r v a t i o n s . However, it i s now e v i d e n t t h a t the d e f e c t i s o n l y p a r t i a l , and must be qualified with respect to the substrate i n question (33). C o n s i d e r a t i o n of the s u b s t a n t i a l amount of comparative d a t a now available shows t h a t the c a t i s unable to g l u c u r o n l d a t e small, w a t e r - s o l u b l e s u b s t r a t e s but t h a t the c o n j u g a t i o n of l a r g e r , more lipid-soluble aglycones proceeds i n the same way as in other species• I n a d d i t i o n to the domestic c a t , a number of r e l a t e d carnivores exhibit an i n a b i l i t y t o g l u c u r o n l d a t e a range of a g l y c o n e s , and this defect i s apparently a biochemical c h a r a c t e r i s t i c of the c a t - l i k e c a r n i v o r e s , the F e l o l d e a . S t u d i e s from t h i s l a b o r a t o r y have documented the o c c u r r e n c e of the d e f e c t i n the lion, lynx, c i v e t and genet, but not the hyena ( 3 3 ) .

There a l s o occur intra-species, or s t r a i n , differences i n glucuronidatlon capacity (33). I n man, these include the virtually complete d e f e c t o f the C r i g l e r - N a j j a r syndrome and the much l e s s s e r i o u s G i l b e r t ' s d i s e a s e , w h i l e i n the r a t the Gunn mutant o f the W i s t a r s t r a i n p r o v i d e s an a n i m a l model of certain aspects of the C r i g l e r - N a j j a r syndrome. Outbred W i s t a r rat


1. CALDWELL


populations also contain mutants d e f e c t i v e i n androsterone glucuronidatlon. These i n t r a - s p e c i e s d i f f e r e n c e s a r e i n g e n e r a l i n d i c a t i v e of the o c c u r r e n c e of i n d e p e n d e n t l y r e g u l a t e d forms of UDPGT.


Sulfate

conjugation

In the s u l f a t e c o n j u g a t i o n r e a c t i o n , a h y d r o x y l ( o r o c c a s i o n a l l y an amino) group p r e s e n t i n a xenobiotic i s linked with the sulfate ion giving a highly polar, highly ionized sulfate ester. The i n o r g a n i c s u l f a t e used i n t h i s c o n j u g a t i o n i s f i r s t a c t i v a t e d by conversion to the high energy sulfate donor 3'p h o s p h o a d e n o s i n e - 5 ' - p h o s p h o s u l f a t e (PAPS) ( 3 8 ) , and the t r a n s f e r of s u l f a t e t o the x e n o b i o t i c i s c a t a l y s e d by a sulf©transferase (39). The enzymes of s u l f a t e a c t i v a t i o n and t r a n s f e r a r e a l l l o c a t e d i n the c y t o s o l of l i v e r and o t h e r t i s s u e s . The s u l f o t r a n s f e r a s e s a r e a f a m i l y of enzymes ( 3 9 ) , s e p a r a b l e by ammonium s u l f a t e f r a c t i o n a t i o n i n t o a group c o n j u g a t i n g p h e n o l i c substrates (39) and another group responsible for steroid s u l f a t i o n (40). The phenol s u l f o t r a n s f e r a s e f r a c t i o n c o n t a i n s a t least four d i s t i n c t enzyme forms, s p e c i f i c a l l y c a t a l y s i n g the sulfation of various substrates including phenols, Nhydroxyacetamides and o e s t r o n e . A wide range of x e n o b i o t i c s may undergo s u l f a t i o n , principally those containing hydroxyl groups but also c e r t a i n aromatic amines. Among the h y d r o x y l i e s u b s t r a t e s a r e a l c o h o l s , phenols, c a t e c h o l s and hydroxylamines (see T a b l e I V ) . In g e n e r a l terms, s u l f a t e c o n j u g a t i o n r e p r e s e n t s an a l t e r n a t i v e to glucuronidatlon f o r the metabolism of a wide range of substrates (41). Two p r i n c i p a l f a c t o r s determine the relative extents to which these two r e a c t i o n s may contribute to the m e t a b o l i s m of a p a r t i c u l a r s u b s t r a t e (1) i t s s t r u c t u r a l f e a t u r e s and (2) the dose g i v e n . S u l f a t i o n i s a f e a t u r e of the metabolism of r e l a t i v e l y small, h y d r o p h i l i c s u b s t r a t e s i n which the c e n t r e undergoing conjugation is sterically unhindered, and whose i n t r a c e l l u l a r d i s t r i b u t i o n f a v o u r s the c y t o s o l (41, 42)• It has long been known t h a t the capacity of the sulfate c o n j u g a t i o n mechanism i s l i m i t e d , and t h a t the p r o p o r t i o n of the dose of a compound u n d e r g o i n g s u l f a t i o n f a l l s with increasing dose s i z e ( 4 3 ) . The s u l f a t e mechanism i s thus more prominent a t lower d o s e s . I t i s g e n e r a l l y a c c e p t e d t h a t the low c a p a c i t y of the r e a c t i o n i s due to l i m i t a t i o n s upon the s i z e of the p o o l of PAPS, and perhaps a l s o i n o r g a n i c sulfate, available for conjugation (44). However, w h i l e t h i s may be the case with respect to i n t e r a c t i o n s between compounds b o t h undergoing sulfation, which may be r e v e r s e d by the a d m i n i s t r a t i o n of PAPS p r e c u r s o r s such as c y s t e i n e , c y s t i n e or i n o r g a n i c s u l f a t e (44), it may not u n d e r l y the r e s t r i c t e d c a p a c i t y of the sulfation mechanism f o r compounds g i v e n s i n g l y . The p o o l of inorganic sulfate feeding PAPS s y n t h e s i s i s r e a d i l y r e p l e t e d from other cellular s u l f a t e precursors, and the c a p a c i t y limitation of


11

12


T a b l e IV.


Functional

Types o f Compounds Forming

group

Sulfates

Example

Primary a l c o h o l

Ethanol

Secondary

Butan-2-ol

alcohol

Phenol

Phenol

Catechol

alpha-Methyl-DOPA

Alicyclic

alcohol

Heterocyclic

Dehydroepiandrosterone

alcohol

3-Hydroxycoumarin

Hydroxyamide

N-hydroxy-2-acetamidofluorene

Aromatic

2-Naphthylhydroxylamine

N-oxide

hydroxylamine

Minoxidil

Drawn from r e f . 57


1. CALDWELL


s u l f a t i o n i n s t e a d may w e l l o r i g i n a t e from the k i n e t i c f e a t u r e s of the s u l f o t r a n s f e r a s e s themselves (45)# Although sulfate conjugation i s widely distributed mammals, the p i g has a d e f i c i e n c y i n the s u l f a t i o n of p h e n o l s (42)*

amongst certain


Methylation The t r a n s f e r of m e t h y l groups t o v a r i o u s hydroxyl, thiol and amino f u n c t i o n s , a l t h o u g h common i n the metabolism of endogenous compounds (46), i s o n l y r a r e l y important q u a n t i t a t i v e l y i n the m e t a b o l i s m of xenobiotics (42, 47)* F u n c t i o n a l groups of xenobiotics u n d e r g o i n g m e t h y l a t i o n (42) i n c l u d e p r i m a r y amines ( e . g . amphetamine), s e c o n d a r y amines ( e . g . desmethylimipramine), tertiary amines, (e.g* dlmethylaminoethanol), azaheterocycles (e*g* p y r i d i n e ) , phenols (e*g* 4 - h y d r o x y - 3 , 5 - d i i o d o b e n z o i c acid), c a t e c h o l s (e*g* alpha-methylDOPA) and t h i o l s ( e . g . thiouracil). I n the m a j o r i t y o f t h e s e c a s e s , methylation serves to Increase lipophilicity and reduce p o l a r i t y of the f u n c t i o n a l group i n q u e s t i o n ( 4 2 ) , and thus the f u n c t i o n of m e t h y l a t i o n i s a p p a r e n t l y not s i m p l e f a c i l i t a t i o n of e x c r e t i o n . However, the m e t h y l a t i o n of t e r t i a r y amines and a z a h e t e r o c y c l e s g i v e s q u a t e r n a r y ammonium compounds w i t h markedly i n c r e a s e d water s o l u b i l i t y ( 4 8 ) . In the g r e a t e s t m a j o r i t y of c a s e s , the m e t h y l group t r a n s f e r r e d t o a x e n o b i o t i c d e r i v e s from the n u c l e o t i d e S - a d e n o s y l m e t h i o n i n e (46), but 5 - m e t h y l t e t r a h y d r o f o l i c a c i d may be the m e t h y l group donor t o p r i m a r y and s e c o n d a r y amines i n the brain (49). A variety of N-methyltransferases are known (50) and thiolmethyltransferase has been the subject of much r e c e n t attention (51). X e n o b i o t i c phenols u n d e r g o i n g m e t h y l a t i o n are generally either catechols or phenols w i t h bulky ortho substituents (42). Comparatively l i t t l e i s known about the s p e c i e s d i s t r i b u t i o n of the v a r i o u s m e t h y l a t i o n r e a c t i o n s . C a t e c h o l and N-methylations apparently o c c u r throughout the Mammalia (46) : i n the case of azaheterocycle p y r i d i n e t h e r e o c c u r r e d a t 10 f o l d v a r i a t i o n in the e x t e n t of m e t h y l a t i o n ( 4 8 ) , from the r a b b i t and c a t which a r e e x t e n s i v e m e t h y l a t o r s (up t o 50% of t h e d o s e ) , t o r a t , mouse and man, i n which m e t h y l a t i o n only accounts f o r 2-10% of an a d m i n i s t e r e d dose* I t i s w e l l known t h a t the O - m e t h y l a t i o n of 4hydroxy-3,5-diiodobenzoic a c i d i s f a r more e x t e n s i v e i n primate s p e c i e s than non-primate mammals (52)* Acetylation A wide v a r i e t y of compounds c o n t a i n i n g the p r i m a r y amino group (NH-), i n c l u d i n g amines, amino a c i d s , s u l f o n a m i d e s , h y d r a z i n e s and h y d r a z i d e s , may undergo N - a c e t y l a t i o n i n the body ( 4 2 ) . Although endobiotics may be a c e t y l a t e d a t -OH ( e . g . choline) or -SH ( e . g . Coenzyme A) c e n t r e s , t h e r e a r e no r e p o r t s of s u c h c e n t r e s i n x e n o b i o t i c s b e i n g so c o n j u g a t e d (42, 47)•


14



The a c e t y l group used to form the amide bond w i t h the amino group derives from the u b i q u i t o u s h i g h energy i n t e r m e d i a t e a c e t y l CoA, and the formation of the bond i s c a t a l y s e d by the enzyme Na c e t y l t r a n s f e r a s e (53). The r e a c t i o n appears to i n v o l v e a p i n g pong B i - B i mechanism, i n w h i c h a c e t y l CoA f i r s t a c e t y l a t e s the enzyme, f o l l o w e d by a c e t y l a t i o n of the amine and r e g e n e r a t i o n of the enzyme ( 5 3 ) . Substrates f o r a c e t y l a t i o n may be c o n v e n i e n t l y d i v i d e d i n t o two groups (53), monomorphic (unimodal), as typified by paraaminobenzoic a c i d and s u l f a n i l a m i d e , and p o l y m o r p h i c (bimodal), s u c h as s u l f a m e t h a z i n e , i s o n i a z i d and dapsone. T h i s nomenclature r e f e r s t o the a b i l i t y of the second group of s u b s t r a t e s to r e v e a l the e x i s t e n c e of two d i s t i n c t forms of the N-acetyItransferase, under s e p a r a t e g e n e t i c ( r e g u l a t o r y ) c o n t r o l , w h i c h c a t a l y s e the a c e t y l a t i o n a t markedly d i f f e r e n t r a t e s . The r a t e of a c e t y l a t i o n is a trait e x h i b i t i n g autosomal Menedelian Inheritance, controlled by a p a i r of a l l e l e s , one f o r f a s t a c e t y l a t i o n (Hf) and one f o r slow (Hs) a c t i n g a t a s i n g l e l o c u s ( 5 4 ) . Thus the population contains t h r e e genotypes, homozygous f a s t (HfUf), homozygous slow (UsHs) and h e t e r o z y g o t e s ( H f H s ) . The phenotyplc e x p r e s s i o n of t h e s e genotypes i s s u c h t h a t the h e t e r o z y g o t e s may only with difficulty be d i s t i n g u i s h e d from homozygous fast acetylators (55). Many attempts have been made to demonstrate the existence of v a r i a n t forms of N - a c e t y l t r a n s f e r a s e in the l i v e r of man and o t h e r s p e c i e s e x h i b i t i n g the polymorphism, but these have been l a r g e l y u n s u c c e s s f u l ( 5 3 ) . Indeed, i t seems l i k e l y t h a t a s i n g l e enzyme a c e t y l a t e s b o t h the 'monomorphic' and ' p o l y m o r p h i c ' s u b s t r a t e s , and t h a t the d i f f e r e n c e between the phenotypes o c c u r r i n g i n p o p u l a t i o n s lies in a differential flexibility of the a c t i v e s i t e . There appears to be an i n d u c e d fit of s u b s t r a t e t o the a c t i v e s i t e , w h i c h i s more f l e x i b l e in the case of f a s t a c e t y l a t o r phenotype, p e r m i t t i n g the a c c e s s of bulkier substrates. The genetic polymorphism controlling N - a c e t y l a t i o n may be demonstrated r e a d i l y i n human p o p u l a t i o n s , where the d i s t r i b u t i o n of the phenotypes shows marked e t h n i c v a r i a b i l i t y ( 5 4 ) . In the U.S.A., the i n c i d e n c e i s c l o s e t o 50% f a s t / 50% slow, w h i l e i n other parts of the world the fast acetylator phenotype predominates up to 90% fast/ 10% slow i n O r i e n t a l s . The polymorphism has a l s o been demonstrated i n outbred animal populations of r a b b i t s and s q u i r r e l monkeys ( 5 6 ) , w h i l e animal models of the f a s t and s l o w a c e t y l a t o r phenotypes occur in v a r i o u s Inbred mouse and hamster s t r a i n s ( 5 6 ) . As w e l l as the g e n e t i c polymorphism of N - a c e t y l a t i o n , t h e r e a r e two marked i n s t a n c e s of s p e c i e s d e f e c t s i n such r e a c t i o n s . The dog (57) and r e l a t e d c a n i n e c a r n i v o r e s , s u c h as the f o x and hyena (57, 58), are u n a b l e to a c e t y l a t e most of the n i t r o g e n c e n t r e s w h i c h a r e s u b s t r a t e s f o r t h i s r e a c t i o n , the e x c e p t i o n b e i n g the alpha-amino group of S - s u b s t i t u t e d c y s t e i n e s and the sulfomanidoN of s u l f o n a m i d e s . Secondly, i t i s well-known t h a t the g u i n e a pig does not e x c r e t e m e r c a p t u r i c a c i d s ( 5 7 ) , a l t h o u g h i t i s a b l e to form g l u t a t h i o n e conjugates ( 5 9 ) , w h i c h a r e the p r e c u r s o r s of


1. CALDWELL


m e r c a p t u r i c a c i d s ( s e e b e l o w ) . The d e f e c t a p p a r e n t l y a r i s e s from an i n a b i l i t y t o N - a c e t y l a t e t h e S - s u b s t i t u t e d c y s t e i n e s formed by catabolism of glutathione conjugates (60). However, the a c e t y l a t i o n of other n i t r o g e n centres i n the guinea p i g occurs i n the same way as most o t h e r mammals ( 5 7 ) .


Glutathione

Conjugation

A l a r g e number o f x e n o b i o t i c s undergo c o n j u g a t i o n with the abundant nucleophilic tripeptide glutathione (gammaglutamyleysteinylglycine), a t i t s f r e e -SH group ( 5 9 , 6 1 ) . The S-substituted glutathiones undergo m e t a b o l i s m by a number of pathways g i v i n g r i s e to various excretory products, t h e most important of which a r e the mercapturic a c i d s ( S - s u b s t i t u t e d Nacetylcysteines). These m a t t e r s w i l l be d e a l t w i t h i n more d e t a i l l a t e r i n t h e c h a p t e r by D r . Bakke. Two d i s t i n c t types o f x e n o b i o t i c s u b s t r a t e s f o r t h e g l u t a t h i o n e conjugation may be d i s c e r n e d ( 5 9 ) , ( a ) those w h i c h are sufficiently e l e c t r o p h i l i c t o undergo c o n j u g a t i o n d i r e c t l y and (b) those which f i r s t undergo m e t a b o l i c a c t i v a t i o n t o produce an e l e c t r o p h i l e which i s then conjugated. The f i r s t group of substrates Includes h a l o - and n i t r o - a l k a n e s and benzenes and sulfonic acid esters, from w h i c h g l u t a t h i o n e d i s p l a c e s an electron-withdrawing group ( 5 9 ) , and a l p h a , beta-unsaturated k e t o n e s and o t h e r compounds w i t h a c t i v a t e d c a r b o n - c a r b o n double bonds e.g. maleic acid diesters. In the l a t t e r cases, g l u t a t h i o n e adds a c r o s s t h e double bond ( 6 2 ) . A v a r i e t y of r e a c t i v e intermediates produced by metabolic o x i d a t i o n may undergo g l u t a t h i o n e c o n j u g a t i o n , I n c l u d i n g o x i r a n e s (arene o x i d e s and a l i p h a t i c and a l i c y c l i c e p o x i d e s ) and r e a c t i v e N-oxidation products. Glutathione opens t h e h i g h l y s t r a i n e d oxlrane ring i n an a d d i t i o n r e a c t i o n (59), or attacks an electron-deprived centre i n the molecule. In addition, glutathione can a l s o i n t e r a c t with metabolically-formed free r a d i c a l species (63). These v a r i o u s g l u t a t h i o n e c o n j u g a t i o n s a r e c a t a l y s e d by a f a m i l y of glutathione S-transferase isozymes (64), located i n the cytosol o f many t i s s u e s . The m u l t i p l i c i t y of t h i s enzyme was r e c o g n i s e d by Boyland and Chasseaud some 20 y e a r s ago ( 6 5 ) , and now some 12 forms have been s e p a r a t e d and c h a r a c t e r i z e d . The number of forms v a r i e s from t i s s u e t o t i s s u e . The g l u t a t h i o n e transferases a r e dimeric p r o t e i n s : a t present seven different s u b u n i t s have been r e c o g n i s e d and the v a r i o u s isozymes a r e homoor h e t e r o - d i m e r s of these ( 6 6 ) . In a d d i t i o n t o c a t a l y s i n g the formation of glutathione conjugates, the glutathione Stransferases are also i n v o l v e d i n the m e t a b o l i s m o f n i t r i t e esters, p e r o x i d e s and a l k y l t h i o c y a n a t e s ( 5 9 ) . I n the case o f the n i t r i t e e s t e r s , a r e a c t i o n which i s s e e m i n g l y a h y d r o l y s i s yielding n i t r i t e and an a l c o h o l i s a c t u a l l y a two s t e p r e a c t i o n , consuming two m o l e c u l e s of g l u t a t h i o n e and g i v i n g r i s e t o the homoconjugate o x i d i z e d g l u t a t h i o n e ( 6 7 ) .


16



Relatively little i s known of the zoological distribution of glutathione conjugation. However, g l u t a t h i o n e S-transferase activity i s v e r y w i d e l y d i s t r i b u t e d throughout mammalian s p e c i e s and t i s s u e s , no i n s t a n c e of a d e f i c i e n c y b e i n g r e c o r d e d ( 5 9 ) . As noted above, the l s o z y m i c make-up of p a r t i c u l a r t i s s u e s may be highly variable (68). This extensive distribution of these enzymes i s p r o b a b l y a r e f l e c t i o n of t h e i r i n c r e a s i n g l y r e c o g n i z e d role i n the metabolism of endogenous s u b s t r a t e s s u c h as s t e r o i d s and l e u k o t r l e n e s ( 6 8 ) , and as a 'defence mechanism' a g a i n s t l i p i d peroxides (63). There i s no p r o p e r l y documented i n s t a n c e of the e x c r e t i o n of a glutathione conjugate i n the u r i n e , a l t h o u g h these m e t a b o l i t e s are found intact i n the b i l e (69). This latter route of elimination i s f a v o u r e d by t h e i r m o l e c u l a r w e i g h t and polarity (70). The urinary excretion p r o d u c t s which d e r i v e from glutathione c o n j u g a t e s a r e numerous, and a l l stem from the Ssubstltuted cysteine produced by the a c t i o n of gamma-glutamyl t r a n s f e r a s e and a p e p t i d a s e , s u c c e s s i v e l y removing the gammaglutamyl and g l y c i n e moieties (71). The p r i n c i p a l routes of m e t a b o l i s m of the S - s u b s t i t u t e d c y s t e i n e s a r e ( a ) N - a c e t y l a t i o n , g i v i n g mercapturic acids (71), and (b) the s o - c a l l e d beta-lyase pathway, i n w h i c h the c y s t e i n e c o n j u g a t e i s c l e a v e d so as t o g i v e rise t o a t h i o l group i n the x e n o b i o t i c t o g e t h e r w i t h , pyruvate and ammonia (72). These t h i o l s a r e then m e t h y l a t e d and the t h i o m e t h y l c o n j u g a t e s , t o g e t h e r w i t h the c o r r e s p o n d i n g s u l f o x i d e s and suIfones produced by subsequent o x i d a t i o n , a r e found i n the excreta and tissues (13, 73). This pathway i s unique in introducing a s i m p l e s u l f u r f u n c t i o n a l group i n t o a xenobiotic, albeit by a r a t h e r roundabout r o u t e . Finally, S-substituted cysteines may undergo t r a n s a m i n a t i o n y i e l d i n g the corresponding thiopyruvic a c i d s , which a r e f u r t h e r m e t a b o l i s e d to thiolactic and t h i o a c e t i c ( t h i o g l y c o l i c ) a c i d s ( 7 4 ) . Of these pathways, the formation of mercapturic acids i s q u a n t i t a t i v e l y the most important, but the b e t a - l y a s e pathway i s now realised to be toxicologically h i g h l y s i g n i f i c a n t i n a number of cases. The d e f e c t of c y s t e i n e c o n j u g a t e N - a c e t y l a t i o n i n the g u i n e a p i g has been n o t e d above. The g l u t a t h i o n e c o n j u g a t i o n mechanism i s of g r e a t s i g n i f i c a n c e i n protecting the body a g a i n s t the h a r m f u l e f f e c t s of e l e c t r o p h i l e s (53) and r a d i c a l s p e c i e s ( 6 3 ) . In a d d i t i o n , i t f a c i l i t a t e s the e x c r e t i o n of many x e n o b i o t i c s , s i n c e most of the p r o d u c t s of the c a t a b o l i s m of c o n j u g a t e s a r e h i g h l y p o l a r and water s o l u b l e ( 7 3 ) . However, i n some cases the c o n j u g a t e s themselves or c e r t a i n of their breakdown p r o d u c t s may be I n v o l v e d i n the expression of t o x i c i t y (56, 73). Epoxide

hydration

One important group of s u b s t r a t e s f o r g l u t a t h i o n e c o n j u g a t e are oxiranes, w h i c h a r e produced by the o x i d a t i o n of a r o m a t i c rings or alkenes. Arene oxides g e n e r a l l y undergo spontaneous rearrangement v i a the 'NIH s h i f t ' mechanism to g i v e phenols ( 7 5 ) , but b o t h they and the a l k e n e o x i d e s may a l s o be hydrated to



CALDWELL


d i h y d r o d i o l s , by the a c t i o n of the enzyme e p o x i d e h y d r o l a s e (76)* A great number of o x l r a n e s a r e s u b s t r a t e s for this reaction, ranging from s i m p l e compounds l i k e s t y r e n e 7,8-oxide and benzene 1,2-oxide t o very complex s t r u c t u r e s s u c h as the various benzo(a)pyrene o x i d e s * Two d i s t i n c t e p o x i d e h y d r o l a s e s e x i s t i n the cell (77), one i n the microsomes (smooth endoplasmic r e t i c u l u m ) and one i n the c y t o s o l * They e x h i b i t differential i n d u c t i o n and i n h i b i t i o n and i n g e n e r a l show d i f f e r e n t , c l e a r - c u t substrate specifities. The c y t o s o l i c enzyme shows a marked preference f o r a l i p h a t i c e p o x i d e s , e s p e c i a l l y those on fatty acids, but i s poor a t h y d r a t i n g the arene oxides typically produced by the o x i d a t i o n of xenobiotics (77)* Phylogenetic d i f f e r e n c e s i n the a c t i v i t i e s of t h e s e two enzymes a r e documented : the microsomal enzyme i s most a c t i v e i n the rhesus monkey and l e a s t a c t i v e i n the mouse, whereas the c y t o s o l i c enzyme i s most active i n the mouse and r a b b i t and l e a s t a c t i v e i n the r a t (77)* However, the i n v i v o s i g n i f i c a n c e of t h e s e d i f f e r e n c e s remains t o be d i s c e r n e d * The

amino a c i d

conjugations

Many x e n o b i o t i c c a r b o x y l i e a c i d s undergo c o n j u g a t i o n w i t h one of a variety of amino a c i d s , i n w h i c h the c a r b o x y l group of the xenobiotic i s l i n k e d i n an amide ( p e p t i d e ) bound w i t h the a l p h a amino group of the amino a c i d (78)* The c h e m i c a l c l a s s e s of a c i d i n v o l v e d i n amino a c i d c o n j u g a t i o n a r e r e l a t i v e l y few i n number, and the r e a c t i o n s a r e r e s t r i c t e d t o c e r t a i n a l i p h a t i c , a r o m a t i c , heteroaromatic, cinnamic and a r y l a c e t i c acids (78)* The occurrence of the r e a c t i o n i s markedly dependent on the steric h i n d r a n c e around the c a r b o x y l group by s u b s t i t u e n t s on the aryl moiety or the s i d e c h a i n b e a r i n g the a c i d f u n c t i o n ( 7 9 ) • The major amino a c i d c o n j u g a t i o n s i n v o l v e glycine, glutamine, taurine and ornithine (56), while i s o l a t e d instances of c o n j u g a t i o n w i t h a number of o t h e r amino a c i d s have been r e p o r t e d (56), including glutanic acid, serine, h i s t i d i n e , a l a m i n e and aspartic acid* In a d d i t i o n t o t h e s e i n s t a n c e s of the c o n j u g a t i o n of a s i n g l e amino a c i d , t h e r e a r e examples of the f o r m a t i o n of d l p e p t l d e conjugates i n v o l v i n g g l y c y l g l y c i n e , g l y c y l t a u r i n e and g l y c y l v a l i n e (80). The p a r t i c u l a r amino a c i d used i n the c o n j u g a t i o n of an a c i d i s a function of i t s structure and the animal species under consideration (80). For benzoic, heterocyclic and cinnamic acids, most s p e c i e s use g l y c i n e , w h i c h i s r e p l a c e d by o r n i t h i n e in b i r d s s u c h as the c h i c k e n ( g a l l l f o r m ) and duck (anseriform). For a r y l - and aryloxy-acetic acids, subprimate mammals use glycine, but t h i s i s r e p l a c e d by g l u t a t a m i n e i n p r i m a t e s . In addition, these acids are conjugated w i t h t a u r i n e , a reaction generally found at a low l e v e l but which i s e s p e c i a l l y well developed i n carnivores* Nature of i n t e r s p e c i e s d i f f e r e n c e s D u r i n g the

c o u r s e of e v o l u t i o n ,

i n xenobiotic

conjugation

l i v i n g organisms have

developed



18


an immense d i v e r s i t y * The Mammalia a l o n e comprise over 4000 species* I t i s remarkable t o note t h a t the b l p h a s i c p a t t e r n of drug m e t a b o l i s m I s found to o c c u r i n a l l mammals (and a l s o most o t h e r o r g a n i s m s ) : however, t h e r e o c c u r widespread q u a l i t a t i v e and q u a n t i t a t i v e v a r i a t i o n s w i t h i n t h i s fundamental p a t t e r n ( 9 , 57), and t h e s e a r e documented a t l e n g t h i n the drug metabolism l i t e r a t u r e Q u a l i t a t i v e d i f f e r e n c e s between s p e c i e s may a r i s e i n one of two ways (a) a s p e c i e s may be (relatively) defective i n a reaction of o t h e r w i s e widespread o c c u r r e n c e , or (b) r e a c t i o n s being restricted i n t h e i r occurrence to p a r t i c u l a r species or groups of s p e c i e s * Quantitative species differences arise from variations i n the r e l a t i v e a c t i v i t i e s of two or more a l t e r n a t i v e m e t a b o l i c o p t i o n s which a g i v e n compound may undergo* It i s this l a s t case w h i c h i s encountered most f r e q u e n t l y * A number of s o - c a l l e d ' s p e c i e s d e f e c t s ' of m e t a b o l i c conjugation have been documented, and a l i s t i s p r e s e n t e d i n T a b l e V . The best known of these examples are the defects of g l u c u r o n i d a t l o n i n the c a t and r e l a t e d f e l i n e s p e c i e s and of Na c e t y l a t i o n i n the dog* I t i s important to a p p r e c i a t e that these d e f e c t s a r e not a b s o l u t e , but must be q u a l i f i e d w i t h r e f e r e n c e to the substrate(s) i n question* Thus, the c a t i s unable to glucuronldate simple, relatively water-soluble phenols and carboxylie acids, but conjugation of more complex, lipidsoluble s u b s t r a t e s proceeds i n the c a t t o the same e x t e n t as in other species* Similarly, dogs a r e unable to N-acetylate aromatic amino groups and h y d r a z i d e s , but do a c e t y l a t e the j>substituted c y s t e i n e s w h i c h a r e the p e n u l t i m a t e i n t e r m e d i a t e s i n the c o n v e r s i o n of g l u t a t h i o n e c o n j u g a t e s t o m e r c a p t u r i c acids* Guinea p i g s , on the o t h e r hand, a p p a r e n t l y cannot N-acetylate t h e s e s u b s t i t u t e d c y s t e i n e s , but do a c e t y l a t e a v a r i e t y of o t h e r amines • For many y e a r s , t h e r e has been much i n t e r e s t i n the p o s s i b i l i t y that certain metabolic r e a c t i o n s have been r e s t r i c t e d by evolutionary pressures to s p e c i f i c groups of species* In particular, the close similarities between man and primate species has l e d t o numerous comparative m e t a b o l i c s t u d i e s w h i c h have thus f a r r e v e a l e d the e x i s t e n c e of five conjugation reactions w h i c h o n l y o c c u r i n these s p e c i e s * These a r e ( i ) the g l u t a m l n e c o n j u g a t i o n of a r y l a c e t i c and a r y l o x y a l k y l a c i d s ( f o u n d in man, apes and O l d and New World monkeys), ( i i ) the 0m e t h y l a t i o n of 4-hydroxy-3,5-diiodobenzoic a c i d (man, O l d and New World monkeys) (52) ( i l l ) the N - g l u c u r o n i d a t l o n of certain methoxysulfonamides ( a l l primates) (33), ( i v ) the q u a t e r n a r y Ng l u c u r o n i d a t i o n of t e r t i a r y a l i p h a t i c amines (man and apes only) (33) and ( v ) the C - g l u c u r o n i d a t i o n of p y r a z o l o n e r i n g s (man and apes o n l y ) (33)* Another example of a m e t a b o l i c r e a c t i o n b e i n g largely restricted i n i t s o c c u r r e n c e t o a group of species i s t h a t of t a u r i n e c o n j u g a t i o n of a r y l a c e t i c a c i d s , which o c c u r s a t low levels i n many s p e c i e s but i s p a r t i c u l a r l y well-developed only i n c a r n i v o r e s (80)* Quantitative

species

differences

i n the

relative

extents


of

1. CALDWELL



competing m e t a b o l i c o p t i o n s can be seen i n the cases of phenols, w h i c h may undergo e i t h e r sulfation or g l u c u r o n i d a t l o n , and carboxylic a c i d s , which are conjugated w i t h amino a c i d s or glucuronic acid* T a b l e VI i l l u s t r a t e s t h i s i n the case of phenol i t s e l f (57)* A major r e a s o n f o r the s t u d y of s p e c i e s d i f f e r e n c e s i n x e n o b i o t i c metabolism i s the interpretation of species differences i n pharmacological and t o x i c responses to c h e m i c a l s * In a d d i t i o n , such d a t a may be of v a l u e i n the z o o l o g i c a l classification of animals, f o r which the term 'pharmacotaxonomy' was c o i n e d (58)* The occurrence of two c h a r a c t e r i s t i c defects i n conjugation reactions i n carnivorous s p e c i e s has been mentioned earlier, those of g l u c u r o n i d a t l o n i n the c a t s and of N - a c e t y l a t i o n i n dogs* Comparative s t u d i e s of s u i t a b l e probe s u b s t r a t e s i n a range of c a r n i v o r e s have p r o v i d e d i m p o r t a n t i n f o r m a t i o n about the t r u e c l a s s i f i c a t i o n of the hyena, an a n i m a l g e n e r a l l y c o n s i d e r e d by z o o l o g i s t s as feline* Table VII presents data on the glucuronidatlon of 1 - n a p h t h y l a c e t i c a c i d and N - a c e t y l a t i o n of sulfadimethoxine i n s i x c a r n i v o r e s , and t h i s shows t h a t the f e l i n e species (cat, l i o n , lynx, c i v e t ) are completely d i s t i n c t from the c a n i n e s (dog and hyena)* The s t a t u s of t h e hyena w i t h r e s p e c t t o these two b i o c h e m i c a l 'marker r e a c t i o n s ' of c a r n i v o r e s strongly suggest that t h i s species should be regarded as a c a n i n e , r a t h e r than a f e l i n e * B i o l o g i c a l s i g n i f i c a n c e o f the m e t a b o l i c c o n j u g a t i o n r e a c t i o n s The v a r i o u s m e t a b o l i c c o n j u g a t i o n r e a c t i o n s of drugs and other chemicals are of g r e a t importance for their biological properties, and reasons f o r t h i s a r e p r e s e n t e d i n T a b l e 8. The majority of c o n j u g a t e s a r e more p o l a r and have markedly greater water s o l u b i l i t y than t h e i r p a r e n t compounds, so t h a t they r e s u l t in both the l o s s of s p e c i f i c r e c e p t o r i n t e r a c t i o n s and facile e l i m i n a t i o n from the body. However, t h e r e o c c u r I n s t a n c e s where e i t h e r the d e t o x i c a t l o n f u n c t i o n of c o n j u g a t i o n may f a i l , either as a r e s u l t of a s p e c i e s d e f e c t or by s a t u r a t i o n , or a c o n j u g a t e c o n t r i b u t e s to b i o l o g i c a l a c t i v i t y . I n a d d i t i o n , the c o n j u g a t i o n r e a c t i o n s have a number of p h a r m a c o k i n e t i c i m p l i c a t i o n s * The c a p a c i t y of the p r i n c i p a l m e t a b o l i c c o n j u g a t i o n s depends upon the a f f i n i t i e s of the t r a n s f e r a s e enzymes i n v o l v e d b o t h f o r the xenobiotic s u b s t r a t e and the endogenous c o n j u g a t i n g agent, and upon the a v a i l a b i l i t y of the c o n j u g a t i n g agent, w h i c h may be limited* T h i s , together w i t h the widespread distribution of c o n j u g a t i o n a c t i v i t y amongst the t i s s u e s of the body ( n o t a b l y i n the a b s o r p t i v e and e x c r e t o r y organs) leads to a number of pharmacokinetic consequences of c o n j u g a t i o n w h i c h a r e l i s t e d in T a b l e V I I I . Space does not permit more than a b r i e f mention of t h e s e , but f u l l d e s c r i p t i o n s a r e t o be found elsewhere* Although these matters a r e l a r g e l y o u t s i d e the scope of t h i s review, they a r e covered i n d e t a i l elsewhere (42, 82, 83)• Species v a r i a t i o n s i n metabolic conjugation, notably o r i g i n a t i n g from s p e c i e s d e f e c t s , a r e of g r e a t s i g n i f i c a n c e as determinants


19


20

T a b l e V.

Some " S p e c i e s D e f e c t s " i n M e t a b o l i c C o n j u g a t i o n

Reaction

Affected species (reference)

Glucuronidatlon

Cat and r e l a t e d s p e c i e s (33)

N - A c e t y l a t i o n of aromatic


Reactions

amines

Dog

and r e l a t e d s p e c i e s (57)

N-Acetylation of S-substituted cysteines

Guinea

Sulfation

P i g (42)

Glutamine c o n j u g a t i o n o f arylacetic acids

Non-primates (80)

G l y c i n e c o n j u g a t i o n of s a l i c y l a t e (but n o t benzoate)

Horse (81)

Table VI.

p i g (57)

S p e c i e s V a r i a t i o n s i n Competing C o n j u g a t i o n The F a t e o f Phenol % dose c o n j u g a t e d w i t h Sulfate Glucuronide

Species

Options:

R a t i o S/G

Man and O l d World monkeys

80

12

7

New World monkeys

25

50

0.5

Rat and mouse

45

40

1

Cat

93

1

93

Pig

2

95

a dapted

from r e f .

5£


< 0.1

CALDWELL


T a b l e V I I . Comparative G l u c u r o n i d a t i o n and A c e t y l a t i o n of V a r i o u s S u b s t r a t e s Amongst C a r n i v o r e s


Glucuronidation of 1-Naphthylacetic acid

N-Acetylation of sulfadimethoxine

Cat

0

18

Lion

0

48

Lynx

0

Civet

0

66

Hyena

40

0

Dog

20

0

T a b l e V I I I . B i o l o g i c a l S i g n i f i c a n c e o f the C o n j u g a t i o n

Reactions

They r e s u l t i n :

1.

Readily excreted

2.

Metabolic a c t i v a t i o n , i n c e r t a i n

3.

D e t o x i c a t l o n , w h i c h may however be d e f e c t i v e , due t o : (a) s p e c i e s

end-products o f x e n o b i o t i c m e t a b o l i s m . cases.

defects

(b) s a t u r a t i o n ( c a p a c i t y l i m i t a t i o n s ) 4.

Pharmacokinetic (a) c a p a c i t y

Implications, including : limitations

(b) e n t e r o h e p a t i c (c) presystemic

recirculation

elimination

(d) d e t e r m i n a t i o n

of r o u t e - o f - e l i m i n a t i o n

(e) drug-drug i n t e r a c t i o n s



22


of biological response. Cats a r e f a r more s u s c e p t i b l e t o the t o x i c e f f e c t s of many g l u c u r o n i d o g e n i c s u b s t r a t e s than a r e rats, rabbits and o t h e r s p e c i e s ( 5 6 ) * A r o m a t i c amines g i v e s r i s e to methaemoglobinaemia i n the dog f a r more e a s i l y than i n other c s p e c i e s which a r e a b l e to N - a c e t y l a t e these compounds* In p l a c e of t h i s c o n j u g a t i o n , dogs a r e a b l e t o a c t i v a t e t h e s e amines by Noxidation (9). N-Acetylation of c a r c i n o g e n i c a r o m a t i c amines has a major sited i r e c t i n g I n f l u e n c e upon t h e i r t u m o r i g e n i c i t y ( 9 ) . I n the dog, these amines a r e p o t e n t b l a d d e r c a r c i n o g e n s , w i t h l i t t l e or no e f f e c t on o t h e r organs, but i n the mouse, r a t and o t h e r s p e c i e s in w h i c h they a r e a c e t y l a t e d , they produce tumours a t m u l t i p l e sites, with l i t t l e or no e f f e c t on the u r i n a r y b l a d d e r * There exist complex inter-relationships between the oxidation, a c e t y l a t i o n , s u l f a t i o n and g l u c u r o n i d a t l o n of a r o m a t i c amines and their c a r c i n o g e n i c i t y , but the N - a c e t y l a t i o n d e f e c t i n the dog has a major r o l e i n d e t e r m i n i n g the t a r g e t f o r c a r c i n o g e n i c i t y * A l t h o u g h c o n j u g a t e s a r e g e n e r a l l y d e v o i d of b i o l o g i c a l activity, for the reasons s t a t e d above, t h e r e a r e a number of welldocumented examples of conjugative metabolites contributing towards the a c t i o n s of the parent compounds* Three c l a s s e s of active conjugates may be d i s c e r n e d (a) s t a b l e c o n j u g a t e s with activity at defined receptor s i t e s , (b) c o n j u g a t e s a c t i n g as reactive i n t e r m e d i a t e s , and ( c ) c o n j u g a t e s a c t i v e a f t e r f u r t h e r metabolism, and important examples of e a c h may be cited (9). Thus, p r o d u c t s of g l u c u r o n i d a t l o n , a c e t y l a t i o n and methylation reactions may have r e c e p t o r a c t i v i t y : i n some c a s e s , the metabolites may become drugs i n their own right e.g. Aacetylprocalnamide (9). The c o n t r i b u t i o n of s u l f a t i o n t o the metabolic activation of N-hydroxyarylamines and 1'hydroxallylbenzenes i s w e l l documented (5, 9, 84), while the f o r m a t i o n of e p i s u l f o n i u m i o n s from the g l u t a t h i o n e c o n j u g a t e s of d i h a l o a l k a n e s u n d e r l i e s t h e i r chemical r e a c t i v i t y (85)* Various examples a r e t o be found i n the l i t e r a t u r e of c o n j u g a t e s giving r i s e t o a c t i v e or r e a c t i v e p r o d u c t s upon f u r t h e r metabolism* One of the b e s t known of t h e s e i s the a n t i t u b e r c u l a r drug isoniazid, whose h e p a t o x i c i t y depends upon the m e t a b o l i s m of i t s N - a c e t y l c o n j u g a t e ( 9 ) . The f u r t h e r m e t a b o l i s m of g l u t a t h i o n e c o n j u g a t e s , normally thought of as d e t o x i c a t l o n products, may similarly result i n the f o r m a t i o n of r e a c t i v e i n t e r m e d i a t e s , as in the cases of h e x a c h l o r o b u t a d l e n e and d i c h l o r o v i n y l c y s t e i n e ( 8 6 ) , or of m e t a b o l i t e s whose a c c u m u l a t i o n l e a d s t o t o x i c i t y : this is well i l l u s t r a t e d by the v a r i o u s m e t h y l s u l f o x i d e s and s u l f o n e s of p o l y h a l o g e n a t e d a r o m a t i c compounds ( 7 3 ) * Concluding

remarks

It w i l l be c l e a r t o the r e a d e r from the above s e c t i o n s t h a t the c o n j u g a t i o n r e a c t i o n s a r e of g r e a t importance i n the metabolic disposition of x e n o b i o t i c s of a l l types In the animal body* These r e a c t i o n s a r e of h i s t o r i c a l s i g n i f i c a n c e i n t h i s f i e l d , but are nowadays i n c r e a s i n g l y r e a l i z e d as h a v i n g p h a r m a c o l o g i c a l and toxicological implications* Any attempt t o r e l a t e t o g e t h e r the


1.

CALDWELL


23


chemical structure, metabolic fate and biological actions of a chemical must take conjugative metabolism into account* The conjugation reactions constitute the second part of the blphasic sequence of xenobiotic metabolism In the body* Although the general features of this sequence are the same throughout mammals (and most other living organisms), the details are highly variable between species* Many examples of quantitative and qualitative differences in metabolic conjugation between species have been illustrated, and these are frequently of functional significance for the toxicity of substrates* By using endogenous conjugation agents, which have well-defined roled in biosynthesis and intermediary metabolism, the conjugation reactions represent an Interface between the metabolism of xenobiotics and the biochemistry of endogenous compounds* The limited supply of certain conjugating agents underlies the ready saturability of some conjugating reactions, but the broader implications of the relationships at this interface remain to be discerned* Since the discovery of the hippuric acid synthesis some 145 years ago, the conjugation reactions have proved a rewarding field of study for successive generations, and continue to generate much interest* In the future, our awareness of these reactions may be expected to develop in various ways (a) enhanced knowledge of both the major and the novel reactions, (b) discovery of more novel reactions, (c) further recognition of the biological consequences of conjugation, and (d) development of the concept of the conjugation reactions as interfaces between xenobiotic and endobiotic biochemistry. Prospects in many of these areas are to be found throughout the present volume. Acknowledgments I am grateful to Professor R. L. Smith for helpful discussion, and to Ms. Irene Ross for the preparation of the typescript. Literature Cited 1.

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25


27. Smith, R.L.; Williams, R.T. In 'Metabolic conjugation and Metabolic Hydrolysis'; Fishman, W.H., Ed.; Academic : New York, 1970; Chap. 1. 28. Jakoby, W.B., Ed. 'Enzymatic Basis of Detoxication, Volume 2' Academic : New York, 1980. 29. Jakoby, W.B.; Ed. 'Methods in Enzymology Academic : New York, 1981.

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56.

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28


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79.

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