The Use of Animal Subcellular Fractions to Study ... - ACS Publications

6 The Use of Animal Subcellular Fractions to Study Type II Metabolism of Xenobiotics MAUREEN

J. C R A W F O R D and D A V I D H . H U T S O N

Shell Research Ltd., Shell Toxicology Laboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent, M E 9 8AG, U . K .

Type II biotransformations or conjugation reactions are enzyme -catalysed energy-requiring biosyntheses. The ultimate reaction requires a high-energy donor substrate, an acceptor substrate and an appropriate transferase. The high-energy substrate may contain the endogenous conjugating agent (conjugand) (e.g. glucuronic acid, as in UDPGA) or i t may contain the xenobiotic (e.g. 4-chlorobenzoic acid, as in 4-chlorobenzoyl-CoA). If the xenobiotic is to be activated to become a donor substrate, other enzymes and high energy substrates (e.g. ATP) are called into action. If we assume for the moment that the low molecular weight substrates for these reactions have equal access to every component in the cell, then the subcellular location of the transferring enzyme will be the controlling factor in the subcellular distribution of the conjugation reaction. History of the Technique The metabolism of foreign compounds has been studied in various subcellular fractions from about 1950. Brodie and coworkers (1) presented the first overview of the enzyme -catalysed metabolism of these compounds in 1958. In the intervening years, the properties of microsomes and other subcellular fractions in relation to the metabolism and toxicity of drugs, pesticides and, more recently, 'environmental' chemicals have received an enormous amount of study. However, i t is interesting to note that the best of the earlier reviews of the enzymology of foreign compound metabolism by J. R. Gillette in 1963 (2) contains the essential details of each of the conjugation reactions referred to below. Progress has not been even: i t has proved difficult to keep our treatment of glucuronyltransferase within reasonable limits, yet amino acid conjugation, despite some very interesting species differences, has received very little attention at the enzyme level. For our practical purposes, the animal cell can be regarded as composed of nucleus, endoplasmic reticulum, mitochondria 0-8412-0486-l/79/47-097-181$12.25/0 © 1979 A m e r i c a n C h e m i c a l Society

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and lysosomes suspended together i n water c o n t a i n i n g d i s s o l v e d p r o t e i n s and small molecules. One o f the values o f studying type I I r e a c t i o n s at the s u b c e l l u l a r l e v e l l i e s i n the informa t i o n gained about the r o l e of these components and the mechanism of the r e a c t i o n s . When the c e l l i s fragmented and the v a r i o u s o r g a n e l l e s are separated, the conjugation r e a c t i o n under i n v e s t i g a t i o n may not be observed i n any o f the f r a c t i o n s because enzymes have been separated from mandatory c o f a c t o r s . For example, g l u c u r o n i d a t i o n r e a c t i o n s are not observed i n the i s o l a t e d endoplasmic r e t i c u l u m (which contains the t r a n s f e r a s e ) because the high-energy donor s u b s t r a t e , u r i d i n e diphosphog l u c u r o n i c a c i d (UDPGA), i s l o c a t e d i n the s o l u b l e f r a c t i o n . Thus the e f f o r t r e q u i r e d to r e c o n s t i t u t e the conjugation r e a c t i o n s at the s u b c e l l u l a r l e v e l has been v i t a l to the f u l l understanding of t h e i r mechanisms. None of t h i s could have happened without the e f f o r t s of biochemists who have developed an understanding of intermediary metabolism and s u b c e l l u l a r f r a c t i o n a t i o n procedures. D i f f e r e n t i a l c e n t r i f u g a t i o n i s s t i l l the main method used and t h e r e f o r e the steady development of p r e p a r a t i v e - s c a l e c e n t r i f u g e s has a l s o been very important. The

Preparation

of S u b c e l l u l a r F r a c t i o n s

S u b c e l l u l a r f r a c t i o n a t i o n i s u s u a l l y c a r r i e d out by d i f f e r e n t i a l c e n t r i f u g a t i o n . However, other methods such as p r e c i p i t a t i o n and chromatography have been i n v e s t i g a t e d f o r the i s o l a t i o n of s p e c i f i c f r a c t i o n s . 2.1 D i f f e r e n t i a l C e n t r i f u g a t i o n D i f f e r e n t i a l c e n t r i f u g a t i o n i s by f a r the most widely used technique. I t i s e f f e c t i v e , clean and gentle and although i t could have been d i s p l a c e d at one time by a f a s t e r technique, the a v a i l a b i l i t y of p r e p a r a t i v e u l t r a - c e n t r i f u g e s capable of up to 500,000 g has cut down p r e p a r a t i o n times to a very competitive l e v e l . The machines now a l s o operate at acceptable noise l e v e l s . Fresh t i s s u e i s homogenised i n b u f f e r e d s a l t s o l u t i o n and the r e s u l t a n t homogenate i s c e n t r i f u g e d at about 600 g to remove unbroken c e l l s and c e l l d e b r i s . The supernatant i s then c e n t r i fuged at 8000-10,000 g f o r about 20 min to sediment the mitochondria. Some lysosomes are sedimented at t h i s stage. The supernatant i s then c e n t r i f u g e d at about 200,000 g f o r 20 min to sediment the fragmented endoplasmic r e t i c u l u m (microsomal f r a c t i o n ) together with some lysosomes. The r e s u l t i n g supernatant i s the s o l u b l e f r a c t i o n ( c y t o s o l ) . Each of the p a r t i c u l a t e f r a c t i o n s may be f u r t h e r p u r i f i e d by washing and r e c e n t r i f u g a t i o n . They may be i n d i v i d u a l l y f u r t h e r p u r i f i e d by d e n s i t y gradient c e n t r i f u g a t i o n . The i s o l a t i o n o f microsomes has r e c e i v e d a l o t of a t t e n t i o n from drug metabolism researchers because of the importance of the microsomal mono-oxygenase system i n x e n o b i o t i c metabolism (see preceding Chapter). When

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the r o u t i n e procedure o u t l i n e d above i s c a r r i e d out there i s i n e v i t a b l y some contamination of one f r a c t i o n by another. This i s best seen by c o n s i d e r i n g a f r a c t i o n a t i o n of r a t l i v e r c a r r i e d out i n our l a b o r a t o r y by Wright and coworkers (3) during s t u d i e s on the e f f e c t of the i n g e s t i o n of d i e l d r i n on hepatocytes. The f r a c t i o n a t i o n procedure was monitored by measuring the a c t i v i t i e s of s e v e r a l enzymes i n each f r a c t i o n , i n c l u d i n g s u c c i n i c dehydrogenase ( m i t o c h o n d r i a l ) , and glucose-6-phosphatase and c h l o r f e n vinphos d e a l k y l a s e (microsomal). Some of these r e s u l t s are i l l u s t r a t e d i n Figure 1. Often we r e q u i r e samples of only the microsomal and c y t o s o l i c f r a c t i o n s , i n which case we r o u t i n e l y prepare 20% homogenates i n 0.1 M potassium phosphate b u f f e r pH 7.4, c e n t r i fuge at 10,000 g f o r 20 min and use t h i s supernatant to prepare microsomes (190,000 g/30 m i n / p e l l e t ) and c y t o s o l (supernatant) (4). P r o t e i n concentrations are r e q u i r e d i n order that enzyme s p e c i f i c a c t i v i t i e s can be c a l c u l a t e d . These are measured u s i n g e i t h e r the s e n s i t i v e procedure d e s c r i b e s by Lowry et a l (5) or the simple m o d i f i c a t i o n of the b i u r e t r e a c t i o n d e s c r i b e d by Robinson and Hodgen (6). Precipitation Rapid methods f o r the p r e p a r a t i o n of microsomal f r a c t i o n s have been looked f o r i n recent y e a r s . I s o e l e c t r i c p r e c i p i t a t i o n of l i v e r microsomes from p o s t - m i t o c h o n d r i a l supernatant at pH 5.4 i s a u s e f u l such method (_7) . Mono-oxygenase c h a r a c t e r i s t i c s i n the product compare very w e l l with those of conventional microsomes but the status of the g l u c u r o n y l t r a n s f e r a s e was not reported. These preparations contain about 45% more p r o t e i n than do conventional preparations and t h e i r p o s s i b l e contamina t i o n by c y t o s o l enzymes, such as the g l u t a t h i o n e t r a n s f e r a s e s , r e q u i r e s f u r t h e r i n v e s t i g a t i o n . A calcium i o n sedimentation method was a c c i d e n t l y discovered during a p r e p a r a t i o n of plasma membranes which became contaminated with aggregated microsomes. The method was then developed s p e c i f i c a l l y f o r microsomes (8). Mono-oxygenase c h a r a c t e r i s t i c s were s i m i l a r to those i n conventionally-prepared microsomes but again g l u c u r o n y l t r a n s f e r a s e was not t e s t e d (9)(10). The method has been a p p l i e d to r a t and r a b b i t kidney and lung t i s s u e . Microsomal y i e l d s from the l a t t e r d i f f e r from those obtained by c e n t r i f u g a t i o n i n that there i s a higher y i e l d of p r o t e i n with lower s p e c i f i c a c t i v i t y ^

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Gel F i l t r a t i o n Chromatography Microsomes and c y t o s o l of r a t l i v e r 13,000 g supernatant have been separated by g e l f i l t r a t i o n through Sepharose 2B; the microsomes were c o l l e c t e d i n the e x c l u s i o n volume (Vo) and the c y t o s o l , between Vo and Vt (12). The method has been a p p l i e d to r a t lung with remarkable success g i v i n g microsomes with very high s p e c i f i c a c t i v i t i e s ( f o r o x i d a t i v e r e a c t i o n s ) (13). An example of one of our own separations using t h i s technique i s shown i n Figure 2 (10 ml of 10,000 g supernatant from 40% r a t

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glucose—6— phosphatase

succinic dehydrogenase

I I

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chlorfenvinphos dealkylase

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40-

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Figure 1.

II

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- ϋ Distribution of marker enzymes between subcelluhr fractions

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Protein mg.ml

-1

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15 Fraction number

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25

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cytochrome P450

Haemoglobin

2 Figure 2.

Gel filtration of rat liver 10,000 g supernatant on Sepharose 2B

30'

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l i v e r homogenate a p p l i e d to a 2.5 cm χ 25 cm column run at upward flow, 1 ml/min; 5 ml f r a c t i o n s c o l l e c t e d ) .

4-5°,

Advantages of the S u b c e l l u l a r F r a c t i o n s I n d i v i d u a l s u b c e l l u l a r f r a c t i o n s , supplemented with the appropriate c o f a c t o r s , u s u a l l y a f f o r d information on d i s c r e t e steps i n metabolic pathways i n a way that i s not p o s s i b l e u s i n g animals, organs or c e l l s . The study of c o f a c t o r requirements provides information on the mechanism of each r e a c t i o n , as do studies w i t h i s o l a t e d p u r i f i e d enzymes. The conditions f o r the r e a c t i o n to be studied can be optimised and f u r t h e r metabolism can, i f necessary, be blocked so that an intermediate of i n t e r e s t can be i s o l a t e d . In a converse sense, now that we know something about the mechanisms of the more common conjugation r e a c t i o n s , a study of the s u b c e l l u l a r l o c a t i o n and c o f a c t o r requirements of the b i o t r a n s f o r m a t i o n of a x e n o b i o t i c o f f e r s u s e f u l information on the metabolism of that compound. I t i s important to know each d i s c r e t e step i n the b i o t r a n s f o r m a t i o n of a x e n o b i o t i c because we then have a chance of i d e n t i f y i n g intermediates that may prove hazardous under c e r t a i n circumstances. I t i s important a l s o to know the e f f e c t of t o x i c a n t s on the f u n c t i o n of a p a r t i c u l a r c e l l o r g a n e l l e . This requires the study of s u b c e l l u l a r f r a c t i o n s . Another important advantage of s u b c e l l u l a r techniques i s that the f r a c t i o n s are r e l a t i v e l y easy to prepare and they are reasonably robust provided that c o r r e c t conditions are used. Conditions are described i n some d e t a i l i n the i n d i v i d u a l s e c t i o n s below. Disadvantages of the Technique The d i f f i c u l t i e s that may be experienced i n working with s u b c e l l u l a r f r a c t i o n s are d e t a i l e d below i n the sections on the i n d i v i d u a l conjugation r e a c t i o n s . However, some u s e f u l g e n e r a l i s a t i o n s may be made. The e x t r a p o l a t i o n back to the s i t u a t i o n i n v i v o from s u b c e l l u l a r systems i s a long one. We tend to base our comparisons on enzyme a c t i v i t y measured under conditions favouring good k i n e t i c s (e.g. zero order f o r c o f a c t o r s , r a t e l i n e a r with time and p r o t e i n c o n c e n t r a t i o n s ) . However, i n v i v o , c o f a c t o r s and/or substrate concentration may be r a t e - l i m i t i n g ; p e n e t r a t i o n of substrate to enzyme may be r e s t r i c t e d or f a c i l i t a t e d ; n a t u r a l m o d i f i e r s may be present; i n t e r a c t i o n between metabolic processes may occur and a f f e c t r e a c t i o n r a t e s . The values KJQ (a measure of enzyme-substrate a f f i n i t y ) and V (the c a p a b i l i t y of the enzyme when saturated with substrate) are v a l u a b l e , p a r t i c u l a r l y i n a comparative sense. T h e i r true relevance to a p a r t i c u l a r s i t u a t i o n i n v i v o however i s d i f f i c u l t to assess. M

A

X

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Another t r a p , but of our own making, must be considered. A r a p i d assay system i s very important i n enzyme p u r i f i c a t i o n and i s very convenient i n t i s s u e and species-comparisons. I t a l s o h e l p s , but i s l e s s easy to arrange, i n s t r u c t u r e - a c t i v i t y s t u d i e s . There has been a tendency i n x e n o b i o t i c enzymology f o r convenience to dominate i n the s e l e c t i o n of substates. The newcomer to the science of x e n o b i o t i c metabolism must s u r e l y be puzzled at the c e n t r a l r o l e occupied by £-nitrophenol. Unsuitable or r e s t r i c t e d s u b s t r a t e s e l e c t i o n has l e d to the p e r p e t r a t i o n of some q u i t e unwarranted g e n e r a l i s a t i o n s . In a d d i t i o n c e r t a i n convenient subs t r a t e s have proved u n s u i t a b l e i n a physico-chemical sense and t h e i r use has l e d to some very complex k i n e t i c s dominated by the p r o p e r t i e s of the s u b s t r a t e r a t h e r than those of the enzyme. Another d i f f i c u l t y , or r a t h e r a p o t e n t i a l f a i l u r e , of approaches u s i n g separated s u b c e l l u l a r f r a c t i o n s i s that one may miss an i n t e r a c t i o n between two processes. The most common example of t h i s i s the production of a metabolite by o x i d a t i o n ( i . e . a type I process) followed by i t s conjugation (type I I p r o c e s s ) . The two r e a c t i o n s may be more than simply consecutive. S p e c i f i c examples w i l l be d i s c u s s e d below. Use

i n X e n o b i o t i c Metabolism

Studies

S u b c e l l u l a r f r a c t i o n s have been used e x t e n s i v e l y to study the d i s c r e t e steps i n x e n o b i o t i c metabolism. They are a l s o very u s e f u l i n a comparative sense. Conjugation r e a c t i o n s , l i k e other b i o t r a n s f o r m a t i o n s , may be a f f e c t e d by a number of f a c t o r s . These i n c l u d e s p e c i e s , t i s s u e , sex, s t r a i n , s t r e s s , age, time, chemicals ( i n d u c t i o n , i n h i b i t i o n , a c t i v a t i o n ) and pregnancy. The e f f e c t s of these on a p a r t i c u l a r b i o t r a n s f o r m a t i o n are conv e n i e n t l y s t u d i e d at the s u b c e l l u l a r l e v e l . For example, i f species comparisons i n v i t r o are shown to be v a l i d i n terms of i n v i v o r e s u l t s across a range of experimental animals, they can be u s e f u l l y extended to human biopsy samples, thus f u r n i s h i n g some metabolic data f o r man. The r e l a t i o n s h i p between chemical s t r u c t u r e and metabolism i s a l s o very conveniently i n v e s t i g a t e d in vitro. These v a r i o u s aspects are e x e m p l i f i e d below f o r the i n d i v i dual conjugation r e a c t i o n s . The r e a c t i o n s r e q u i r i n g a c t i v a t e d conjugand (glucuronide formation, s u l p h a t i o n , phosphorylation, a c e t y l a t i o n and methylation) are d i s c u s s e d f i r s t followed by those i n v o l v i n g a c t i v a t i o n of the x e n o b i o t i c (amino a c i d conj u g a t i o n ) . Glutathione conjugation, which depends upon the mutual i n t r i n s i c r e a c t i v i t y of both s u b s t r a t e s , i s d i s c u s s e d l a s t . Conjugation w i t h Glucuronic A c i d Mechanism and l o c a t i o n . Glucuronic a c i d conjugation i s probably the most q u a n t i t a t i v e l y important of the Type I I processes, both i n terms of p r o p o r t i o n of a p a r t i c u l a r x e n o b i o t i c being so conjugated, and i n the v a r i e t y of compounds t a k i n g p a r t


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i n the r e a c t i o n . Phenols, a l c o h o l s , c a r b o x y l i c acids and N-hydroxy compounds a l l form O-glucuronides and a number of S- and Nglucuronides have been detected. The process has r e c e n t l y been reviewed i n some d e t a i l by Dutton and co-workers (14)(15). This process i s an example of one i n which the endogenous conjugand ( g l u c u r o n i c acid) i s a c t i v a t e d to a high energy donor (uridine-5 -diphospho-a-D-glucuronic a c i d , UDPGA). The t r a n s f e r of the α-D-glucopyranuronyl group from UDPGA to the acceptor, forming £-D-glucopyranuronosides, i s c a t a l y s e d by UDPGAglucuronyl transferase (EC 2.4.1.17). The enzyme i s l o c a t e d i n the endoplasmic r e t i c u l u m of mammalian c e l l s and t h e r e f o r e appears, on s u b c e l l u l a r f r a c t i o n a t i o n , i n the microsomes. I s o l a t i o n , p r o p e r t i e s and use. The components r e q u i r e d f o r an i n v i t r o study: microsomes, UDPGA, x e n o b i o t i c and b u f f e r , are r e a d i l y a v a i l a b l e . UDPGA i s a l s o a v a i l a b l e r a d i o l a b e l e d . T h i s simple system i s p e r f e c t l y adequate f o r many s t u d i e s of p e s t i c i d e metabolism. I t i s i n a p p r o p r i a t e to review assay methods i n great d e t a i l i n t h i s chapter because, i n x e n o b i o t i c metabolism, we are so o f t e n i n t e r e s t e d i n a s p e c i f i c compound or s e r i e s of compounds and the assay procedure w i l l be based on the conversion of that compound. There are, however, some recent methods that may be g e n e r a l l y u s e f u l . A method i n v o l v i n g UDP[1^C]GA u t i l i s e s an Amberlite XAD-2 column to separate unchanged UDPGA, conjugate and unchanged substrate (16). A continuous assay based on the enzymatic assay of the UDP r e l e a s e d during the t r a n s f e r has a l s o been reported (17). The i s o l a t i o n of the enzyme i s achieved by the p r e p a r a t i o n of microsomes as described above. The enzyme i s r e l a t i v e l y s t a b l e i n f r o z e n 10,000 g supernatant and i n f r o z e n microsomal p e l l e t . I f s t o r e d at -196°C ( l i q u i d n i t r o g e n ) , r a t and r a b b i t enzymes can be kept f o r many days (18).Because the enzyme i s c l o s e l y a s s o c i a t e d with the l i p o p r o t e i n microsomal membranes, f u r t h e r p u r i f i c a t i o n must be preceded by s o l u b i l i s a t i o n . A recent method (19) i n v o l v e s treatment of the microsomes with 1% Lubrol 12A9 (a condensate of dodecyl a l c o h o l with approx. 9.5 mol of ethylene oxide per mol) f o r 20 min at 4°C. The enzyme remained i n the supernatant a f t e r f u r t h e r c e n t r i f u g a t i o n i . e . s o l u b i l i s a t i o n apparently occurred. I t was then p r e c i p i t a t e d w i t h ammonium sulphate and f u r t h e r p u r i f i e d i n the presence of 0.05% L u b r o l by DEAE c e l l u l o s e chromatography. The product contained only 3 p o l y p e p t i d e s ; enzyme a c t i v i t i e s towards 2-aminophenol and 4 - n i t r o phenol were increased 43- and 4 6 - f o l d r e s p e c t i v e l y . The p u r i f i e d enzyme had much improved s t a b i l i t y i n comparison with the microsomally-bound enzyme. Although there i s much k i n e t i c evidence f o r the existence of s e v e r a l glucuronyl t r a n s f e r a s e s (15), very few p a i r s of aglycone spécificités have been separated p h y s i c a l l y . One such separation i s that of the a c t i v i t i e s towards 4-nitrophenol and morphine by D e l l V i l l a r et a l (20). I t i s i n t h i s area where p u r i f i c a t i o n of the enzymes i s important. P u r i f i c a t i o n , however d e s i r a b l e i n theory and however necessary f

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to the study of the enzyme, i s r a r e l y c a r r i e d out i n s t u d i e s of p e s t i c i d e conjugation. Our main concern i s the f a t e of the p e s t i c i d e i n the whole animal and the r o l e that the conjugation may p l a y i n the d i s p o s i t i o n of the p e s t i c i d e . Nevertheless we have to be aware of some p r a c t i c a l complications consequent on the p a r t i c u l a t e nature of t h i s enzyme. UDPGA-glucuronyl t r a n s f e r a s e i s ' l a t e n t i n f r e s h l y p r e pared microsomes. Membrane-perturbing processes such as aging, f r e e z i n g and thawing, and treatment with detergents, chaotropes, organic s o l v e n t s , a l k a l i , t r y p s i n or phospholipases may a c t i v a t e the enzyme by a f a c t o r of 40 or more (15). T h i s property, together with evidence d e r i v e d from k i n e t i c s t u d i e s , some i n v o l v i n g competitive s u b s t r a t e s , i n d i c a t e s that the enzyme i s deeply b u r i e d , p o s s i b l y on the inner surface of the microsomal v e s i c l e . For t h i s reason i t i s advisable to p r e t r e a t microsomes with T r i t o n X-100 ( o v e r a l l 0.05% s o l u t i o n ) before adding the x e n o b i o t i c s u b s t r a t e . This treatment should f u l l y a c t i v a t e the enzyme and remove e r r o r s due to unknown amounts of a c t i v a t i o n caused by age, storage, mechanical e f f e c t s or even an e f f e c t of the x e n o b i o t i c substrate i t s e l f . The treatment should a l s o allow access of both UDPGA and the x e n o b i o t i c to the enzyme. T h i s may be r e s t r i c t e d , p a r t i c u l a r l y i f an i o n i c substrate (e.g. a c a r b o x y l i c acid) i s being i n v e s t i g a t e d . There are two f u r t h e r problems a s s o c i a t e d with i t s use however. The amount of a c t i v a t i o n i s species-dependent. For example, the g l u c u r o n i d a t i o n of 7-hydroxychlorpromazine (21) i s a c t i v a t e d twice as much i n r a t l i v e r microsomes as i t i s i n guinea-pig l i v e r microsomes. A c t i v a t i o n i s a l s o dependent on the c o n c e n t r a t i o n of T r i t o n X100 (see Figure 3). 1

Another problem i s the d e s t r u c t i o n of UDPGA by pyrophosphorylase. This i s p a r t l y i n h i b i t e d by EDTA but a b e t t e r s o l u t i o n i s apparently the use of c i t r a t e b u f f e r which v i r t u a l l y abolishes the pyrophosphorylase a c t i o n (15). The enzyme has a pH optimum of about 7.4 though t h i s may vary somewhat with i o n i s a b l e s u b s t r a t e s . Reaction rates are l i n e a r f o r at l e a s t an hour at 37°C. Examples of use. One of the most frequent uses of the g l u c u r o n i d a t i o n system i n our l a b o r a t o r y i s i n the b i o s y n t h e s i s of glucuronides of f a e c a l m e t a b o l i t e s . These are then chromatog r a p h i c a l l y compared with suspected glucuronides found i n the u r i n e or i n the b i l e of t r e a t e d animals. Figure 4 i l l u s t r a t e s the formation of the glucuronide of a n t i - 1 2 - h y d r o x y - [ l ^ C l e n d r i n under the f o l l o w i n g c o n d i t i o n s : volume, 5 ml; hydroxyendrin, 0.004 mM; UDPGA, 0.75 mM; washed r a b b i t l i v e r microsomes (4.2 mg/ml) i n 0.1 M TRIS-HC1 b u f f e r (pH 7.4 at 37°C); i n c u b a t i o n temperature, 37°C. P o r t i o n s (1 ml) were withdrawn at i n t e r v a l s and p a r t i t i o n e d between benzene and water which were then r a d i o assayed to a f f o r d the proportions of unchanged substrate and conjugate r e s p e c t i v e l y (22). T r i t o n X-100 had no e f f e c t on t h i s r e a c t i o n , p o s s i b l y because the very l i p o p h i l i c a n t i - 1 2 -


0.1

0.2

0.3

0.4

0.5

Concentration of Triton X-100 (%) ure 3.

Effect of Triton X-100 on glucuronidation: ( ), rat liver microsomes; ( ), guinea pig liver microsomes.

CRAWFORD AND HUTSON

Figure 4.

Type

II

Metabolism

Time course of the glucuronidation of C anti-12-hydroxyendrin by rabbit liver microsomes 14

192


hydroxyendrin was r e a d i l y a c c e s s i b l e to the enzyme. The common experimental animals, e.g. guinea-pig, r a t , mouse, hamster and dog, a l l possess reasonable amounts of h e p a t i c microsomal g l u c u r o n y l t r a n s f e r a s e . L i t t e r s t e t a l (23) have compared the h e p a t i c enzyme i n rhesus monkey ( c u r r e n t l y i n short supply) with species which may be used as a l t e r n a t i v e t e s t animals. A c t i v i t i e s towards 4-nitrophenol (nmol per min per mg p r o t e i n ) were: rhesus monkey, 7; s q u i r r e l monkey, 8-11 (sex d i f f e r e n c e ) ; common t r e e shrew (9-12); miniature p i g (6-9); Sprague-Dawley r a t (2-3). These measurements were made without the a d d i t i o n of detergent. Conditions were: s u b s t r a t e , 0.2 mM; UDPGA, 3.3 mM; p r o t e i n , 1 mg/ml; T r i s b u f f e r pH 7.4, 1 mM. The cat and other F e l i d a e excrete l i t t l e o r no glucuronide conjugates of x e n o b i o t i c s (24). The h i g h K value f o r 4-nitrophenol with cat l i v e r g l u c u r o n y l t r a n s f e r a s e compared to that with r a t l i v e r t r a n s f e r a s e (25) i s i n accord with t h i s o b s e r v a t i o n . Hens s i m i l a r l y do not excrete many glucuronide conjugates and have very low enzyme a c t i v i t i e s i n l i v e r . These are examples of the i n v i t r o s u b c e l l u l a r r e s u l t s comparing w e l l w i t h the s i t u a t i o n i n v i v o . The occurrence of g l u c u r o n y l t r a n s f e r a s e i n b i r d s g e n e r a l l y has not been s t u d i e d . F i s h excrete glucuronides (26) and have been shown to possess h e p a t i c g l u c u r o n y l t r a n s f e r a s e a c t i v i t y towards 2-aminophenol (27) 3 - t r i f l u o r o m e t h y l - 4 - n i t r o phenol (26), and 4-nitrophenol (28). Glucuronide formation i s important i n man but the h e p a t i c enzyme has not been examined in detail. m

Another important use of i n v i t r o techniques i s f o r the comparison of b i o t r a n s f o r m a t i o n i n d i f f e r e n t t i s s u e types. However, a f t e r a survey u s i n g a s e n s i t i v e me thylumb e l l i f e r o n e assay, A i t i o (29) concluded that l i v e r i s the most important organ of glucuronide s y n t h e s i s . I t was estimated that the whole g a s t r o i n t e s t i n a l t r a c t possessed only 15-20% of the enzyme a c t i v i t y found i n l i v e r . Some r e s u l t s are summarised i n Table I. Low enzyme a c t i v i t y has a l s o been found i n the p l a c e n t a of s e v e r a l s p e c i e s , i n c l u d i n g man (30). The r e l a t i v e a c t i v i t i e s found f o r v a r i o u s t i s s u e s are p a r t l y a f u n c t i o n of the choice of s u b s t r a t e . For example, b r a i n , h e a r t , f a t and diaphragm possess very low a c t i v i t i e s towards methylumbelliferone (30) but q u i t e h i g h activités towards the c a r b a r y l m e t a b o l i t e , 1-naphthol (31). This demonstrates that model s u b s t r a t e s serve only as a guide; f o r r e l e v a n t i n f o r m a t i o n on a s p e c i f i c chemical, only s t u d i e s on that chemical w i l l r e a l l y s u f f i c e . T o x i c o l o g i c a l s i g n i f i c a n c e . The value of g l u c u r o n i d a t i o n l i e s i n the dramatic change i n p o l a r i t y that the process confers. The glucuronides are also very r e a d i l y secreted i n b i l e or v i a the kidneys and thus removed from the body. G l u c u r o n i d a t i o n can a l s o prevent c e r t a i n types of metabolite from being f u r t h e r b i o a c t i v a t e d to r e a c t i v e species such as the quinones which may be formed from aromatic d i h y d r o d i o l s (32). The process i s almost always a d e t o x i f i c a t i o n , however i n some important

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cases the reverse i s observed. The g l u c u r o n i d a t i o n o f Ν-hydroxyarylacetamides (e.g. N-hydroxyphenacetin, Figure 5) a f f o r d s chemically r e a c t i v e molecules capable of i n t e r a c t i n g with t i s s u e macromolecules (33). The demonstration of t h i s type o f b i o a c t i v a t i o n i s a valuable use of i n v i t r o t e s t systems.

Table I. Glucuronidation o f methylumbelliferone in rat tissues

Enzyme a c t i v i t y Tissue

Liver Duodenal mucosa Adrenal glands Kidneys Spleen Lungs Thymus Heart Brain

nmol product p e r g wet wt. l i v e r 460 260 170 150 30 28 19 1.4 0.8

nmol product p e r whole organ 3200 96 63 120 35 34 9 1.1 1.3

The use of separated s u b c e l l u l a r f r a c t i o n s may f a i l to r e v e a l p o s s i b l e i n t e r a c t i v e e f f e c t s or f u n c t i o n a l r e l a t i o n s h i p s between the f r a c t i o n s . For example, the a d d i t i o n of UDPGA to r a t l i v e r microsomes increases t h e i r rate of 12-hydroxylation of d i e l d r i n (34). The r a t i o n a l e f o r adding UDPGA t o an o x i d a t i v e system was that sjn-12-hydroxydieldrin (Figure 6), although more h y d r o p h i l i c than d i e l d r i n , i s s t i l l a very l i p o p h i l i c molecule, p a r t i c u l a r l y i n view of the hydrogen bonding of the hydroxy1 group to the epoxide oxygen. Thus, when formed as a metabolite v i a the a c t i o n o f microsomal mon o-oxygen as e, i t would remain near i t s s i t e of formation, p o s s i b l y i n h i b i t i n g f u r t h e r h y d r o x y l a t i o n of d i e l d r i n . This e f f e c t was f i r s t noted by von Bahr and B e r t i l s s o n (35) with demethylimipramine. The f u n c t i o n a l r e l a t i o n s h i p between microsomal monooxygenase, epoxide hydratase and glucuronyl t r a n s f e r a s e has r e c e n t l y been i n v e s t i g a t e d i n l i v e r microsomes and i n i s o l a t e d hepatocytes (32). The r e s u l t s provide a good i l l u s t r a t i o n of one of the l i m i t a t i o n s of the s u b c e l l u l a r approach. In the hepatocyte and i n microsomes f o r t i f i e d with NADPH and UDPGA, naphtha lene i s oxygenated to i t s 1,2-oxide which i s cleaved t o a d i h y d r o d i o l (by epoxide hydratase) which, i n t u r n , i s conjugated with g l u c u r o n i c a c i d (Figure 7). However, microsomes a f f o r d the d i h y d r o d i o l as the major metabolite; hepatocytes a f f o r d the glucuronide. Only by using e i t h e r more microsomal p r o t e i n o r the

194

Figure 5.


Conjugation in the formation of reactive metabolites from phenacetin

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Cl

Type

Cl

Figure 6.

II

195

Metabolism

Cl

Hydroxyhtion and glucuronidation of dieldrin

196

XENOBIOTIC

mo no-oxygenase plus 0

2

METABOLISM

epoxide hydratase + H 0 2

microsomal glucuronyl transferase + UDPGA

Figure 7.

Rehtionship between the enzymes affecting the metabolism of naphthalene

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a l l o s t e r i c e f f e c t o r of g l u c u r o n y l t r a n s f e r a s e , UDP-N-acetylglucosamine, could microsomes be f o r c e d to y i e l d reasonable q u a n t i t i e s of glucuronide. T h i s e f f e c t o r may a l s o c o m p e t i t i v e l y i n h i b i t the d e s t r u c t i v e e f f e c t of the pyrophosphorylase on UDPGA in vitro. Conjugation with Sulphate Mechanism and l o c a t i o n . Conjugation with sulphate i s mediated by the s u l p h o t r a n s f e r a s e enzymes ( h i s t o r i c a l l y sulphok i n a s e s ) . The donor s u b s t r a t e , which contains the a c t i v a t e d sulphate group, i s 3'-phosphoadenosine-S -phosphosulphate (PAPS). Acceptor groups are p r i n c i p a l l y p h e n o l i c but an a l c o h o l i c or primary amine f u n c t i o n can a l s o be sulphated v i a the same mechanism. I r r e s p e c t i v e of the chemical nature of the f i n a l sulphated product, sulphoconjugation f o l l o w s the same general pathway: a c t i v a t i o n of i n o r g a n i c sulphate to y i e l d f i r s t adenosine 5 -phosphosulphate (APS) and then 3 -phosphoadenosine 5 phosphosulphate (PAPS), followed by t r a n s f e r of the sulphate group from PAPS to s u i t a b l e acceptors: 1

1

f

2

ATP

+ S0 ~

*

APS

+ ATP

*

4

PAPS + Acceptor

PAP

APS

f

+ PP£

(1)

PAPS + ADP

(2)

+ sulphated acceptor

(3)

The enzymes c a t a l y s i n g these r e a c t i o n s are (1) ATP-sulphate adenylyl t r a n s f e r a s e (ATP-sulphurylase); (2) ATP-adenylyl sulphate 3 -phosphotransferase (APS-kinase) and (3) an appropriate s u l p h o t r a n s f e r a s e (EC 2.8.2). In mammalian c e l l s the enzymes r e s p o n s i b l e f o r the p r o d u c t i o n of PAPS from sulphate are l o c a l i s e d i n the c y t o s o l . Sulphotransferases are l o c a t e d i n both the microsomal and the c y t o s o l i c f r a c t i o n s of a wide v a r i e t y of t i s s u e s ; t h e i r general t i s s u e d i s t r i b u t i o n resembles that of the UDP-glucuronyltransferases, except that they are a l s o abundant i n p l a c e n t a . Boundaries of t h e i r s p e c i f i c i t i e s are more c l e a r than those of the g l u c u r o n y l t r a n s f e r a s e s , and d i s t i n c t i v e phenol, s t e r o i d and arylamine s u l p h o t r a n s f e r a s e a c t i v i t i e s have been detected. Other t r a n s f e r a s e s deal apparently s o l e l y with endogenous compounds, e.g. cerebroside and p o l y s a c c h a r i d e sulphot r a n s f erases. Simple endogenous sulphates i n c l u d e s those of s t e r o i d s , a d r e n a l i n e , t r i - i o d o t h y r o n i n e and s e r o t o n i n . The p i c t u r e emerges ( c f . the g l u c u r o n y l t r a n s f e r a s e s ) of the sulphot r a n s f e r a s e s r e s p o n s i b l e f o r the s u l p h a t i o n of small molecules being f r e e l y s o l u b l e i n the c y t o s o l , whereas those that are i n v o l v e d i n the assembly of l a r g e r molecules are arranged, together with other r e q u i s i t e b i o s y n t h e t i c enzymes, i n assemblyl i n e f a s h i o n on the membranes of the endoplasmic r e t i c u l u m and the G o l g i apparatus. I t i s extremely d o u b t f u l whether any of these bound enzymes p l a y any r o l e i n the metabolism of xenob i o t i c s (36). f

198


I s o l a t i o n , p r o p e r t i e s and use. The u n c e r t a i n t y i n the t o t a l number, p r e c i s e r o l e s and s p e c i f i c i t i e s of the s u l p h o t r a n s f e r a s e s r e f l e c t s the f a c t that they are exceedingly d i f f i c u l t to p u r i f y and to separate from one another. Moreover, some of them aggre gate and/or change conformation under c e r t a i n c o n d i t i o n s ; m u l t i p l e peaks of s i m i l a r a c t i v i t y , which may o r may not be due to the same enzyme, appear on chromatography columns and are a constant hindrance to the e x p e r i m e n t a l i s t . A s u l p h o t r a n s f e r a s e has been p a r t i a l l y p u r i f i e d from bovine kidney (acetone powder) using 4-nitrophenol as the assay sub s t r a t e (37). The a c t i v i t y of the enzyme was measured using [35s]PAPS £ the assay mixture. Substrate s p e c i f i c i t y was i n v e s t i g a t e d and i t was found to be r e l a t i v e l y s p e c i f i c f o r simple a r y l sulphate formation as shown i n Table I I . n

Table I I . R e l a t i v e substrate s p e c i f i c i t i e s f o r bovine kidney s u l p h o t r a n s f e r a s e

Substrate

Relative specificity

4-Nitrophenol 4-Hydroxybenzaldehyde 4-Chlorophenol 1-Naphthol Phenol o-Cresol m-Cresol 3-Nitrophenol 3-Hydroxybenzaldehyde

100 75.9 79.7 32.5 6.5 21.7 8.3 49.8 21.0

No a c t i v i t y could be demonstrated towards e t h a n o l , propan-Ι οί, b u t a n - l - o l , or towards a range of s t e r o i d s . The i s o l a t i o n of N-hydroxy-2-acetylaminofluorene (N-0H-2AAF) s u l p h o t r a n s f e r a s e has r e c e n t l y been achieved from the c y t o s o l f r a c t i o n s of male and female r a t l i v e r s (the l a t t e r possess very low a c t i v i t y ) (38). A 2000-fold p u r i f i c a t i o n with a y i e l d of over 12% was achieved using the f o l l o w i n g procedure: ammonium sulphate f r a c t i o n a t i o n , DEAE-cellulose column chromatography, hydroxya p a t i t e column chromatography, sephadex G-200 g e l f i l t r a t i o n , i s o e l e c t r i c f o c u s s i n g and, f i n a l l y , more sephadex G-200 g e l f i l t r a t i o n . The f i n a l p r e p a r a t i o n was homogenous on a n a l y t i c a l d i s c g e l e l e c t r o p h o r e s i s . The p u r i f i e d enzyme had a c t i v i t y towards 4-nitrophenol with an approximately 1600-fold i n c r e a s e i n s p e c i f i c a c t i v i t y over the crude homogenate, but i t had very low a c t i v i t y towards endogenous s t e r o i d s and s e r o t o n i n . PAPS was used as the sulphate donor i n these assay mixtures and was synthesised e n z y m a t i c a l l y (39). The pure enzyme was very u n s t a b l e , e s p e c i a l l y i n d i l u t e s o l u t i o n s . T h i o l compounds were found to have a s t a b i l i s i n g e f f e c t and t h i o l b l o c k i n g reagents were potent i n h i b i t o r s .

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The i n v o l v e d nature of these p u r i f i c a t i o n s suggests that the study of phase I I sulphoconjugations u s i n g p u r i f i e d sulphotrans ferase from v a r i o u s animal species and animal t i s s u e s i s impract i c a l at present. An i n v i t r o assay system f o r studying these r e a c t i o n s u s i n g crude s u b c e l l u l a r f r a c t i o n s has been developed by Mulder et a l (33). I t comprises c y t o s o l (600 μg p r o t e i n / m l ) , x e n o b i o t i c (0.5 mM), T r i s - H C l b u f f e r (100 mM, pH 8.0) and a PAPSgenerating system (PAPS-GS): 3 5 - a d e n o s i n e diphosphate (PAP, 10 μΜ) and 4-nitrophenyl sulphate (10 mM). In t h i s assay mixture 4-nitrophenyl sulphate i s used to convert PAP i n t o PAPS, a r e a c t i o n presumably c a t a l y s e d by a phenolsulphotransferase i n the c y t o s o l . The r a t e of formation of 4-nitrophenol i s spectrophotom e t r i c a l l y determined and i s c a l c u l a t e d by s u b t r a c t i n g the amount of 4-nitrophenol r e l e a s e d i n a c o n t r o l i n c u b a t i o n mixture ( x e n o b i o t i c absent) from that r e l e a s e d i n the presence of the x e n o b i o t i c . The r a t e of s u l p h a t i o n may thus be i n d i r e c t l y c a l c u l a t e d from the amount of 4-nitrophenol r e l e a s e d u s i n g a molar e x t i n c t i o n c o e f f i c i e n t of 17,500 M" cm~l (at pH 8.0). This assay procedure makes an important c o n t r i b u t i o n to the f i e l d of xeno b i o t i c metabolism i n that i t provides a cheap and simple method f o r studying the mechanisms and r a t e s of phase I I sulphate con j u g a t i o n r e a c t i o n s using a PAPS-GS and c y t o s o l . H i t h e r t o PAPS has been commercially a v a i l a b l e i n the l a b e l l e d form only and i t s enzymic s y n t h e s i s i s a tedious process (39)(40). A more t r a d i t i o n a l assay procedure i s that described by Wu and Straub (38) where PAPS i s used at a c o n c e n t r a t i o n of approximately 0.3 mM when substrate concentration i s 0.2 mM. f

1

1

Examples of use. Harmol (Figure 8) i s a good substrate f o r phenol sulphotransferase i n r a t l i v e r 600 g f r a c t i o n (41) but harmalol (Figure 8) i s a very poor s u b s t r a t e . The reason f o r the d i f f e r e n c e i n r a t e of s u l p h a t i o n of these two substrates i s unknown, but the f i n d i n g s agree with the d i f f e r e n t r a t e s and modes of conjugation ( s u l p h a t i o n and g l u c u r o n i d a t i o n ) of the two compounds found i n v i v o . The metabolism of harmol i l l u s t r a t e s a problem that o f t e n a r i s e s when d i s c u s s i n g the phase I I conjugation of x e n o b i o t i c s with sulphate. Sulphation of a x e n o b i o t i c or i t s metabolite i s p r a c t i c a l l y always accompanied by conjugation with g l u c u r o n i c a c i d . Glucuronic a c i d conjugation o f t e n predominates and t h i s has been assumed to be due to a l i m i t e d supply of sulphate i n v i v o . Mulder and coworkers have developed a method f o r the simultan eous measurement of UDP-glucuronyltransferase and phenolsulphot r a n s f e r a s e from r a t l i v e r i n v i t r o , using harmol as substrate and 600 g supernatant as the enzyme source (42). T r i t o n X-100 was used to a c t i v a t e g l u c u r o n y l t r a n s f e r a s e ( S e c t i o n 5.1) and was shown to have no e f f e c t on the a c t i v i t y of phenolsulpho t r a n s f erase. The amount of the conjugates formed was measured f l u o r o m e t r i c a l l y and the a c t i v i t i e s of the enzymes were expres sed as a r b i t r a r y u n i t s of fluorescence recovered from a t i c a n a l y s i s of the incubates. At low substrate concentrations

XENOBIOTIC

METABOLISM

glucuronidation sulphation phosphorylation (?)

,οχ COCH

3

(reactive) Figure 9.

Acetyhtion and conjugation in the activation of aromatic amines

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Metabolism

201

( arene oxide -> d i h y d r o d i o l -> catechol monomethylcatechol. Most of the enzyme a c t i v i t y i s present i n the c y t o s o l of v a r i o u s t i s s u e s . Phenol 0-methy1transferase, a microsomal enzyme, has been detected i n v i t r o (68) but i t s s i g n i f i c a n c e i n v i v o i s unknown because the 0-methylation of monohydric phenols apparently does not occur i n v i v o . A p o t e n t i a l l y u s e f u l general assay method i n v o l v i n g the use of the t r i t i a t e d cosubstrate S-adenosy1-L[methyl-3H]methionine has been described (69). The r a d i o a c t i v e methylated product i s e x t r a c t e d i n t o organic solvent f o r r a d i o assay. S-Methylation. ^ - M e t h y l a t i o n i s a general pathway of metabo l i s m of the thiopyrimidone a n t i t h y r o i d drugs such as 6-propyl2 - t h i o u r a c i l . T e t r a h y d r o f u r f u r y l mercaptan i s a l s o S-methylated i n an S-adenosylmethionine-dependent enzymatic r e a c t i o n . The enzyme was found i n the microsomal f r a c t i o n of l i v e r , kidney and small i n t e s t i n e (70). Thiophenol l i b e r a t e d during the metabolism of dyfonate (Figure 11) i s methylated (71). This r e a c t i o n has not been s t u d i e d i n v i t r o . N-Methylation. P y r i d i n e d e r i v a t i v e s are N-methylated but the r e a c t i o n s are q u a n t i t a t i v e l y of minor importance. The most widely s t u d i e d N-methylation r e a c t i o n s are those i n v o l v i n g the b i o g e n i c amines. For example, phenylethanolamine N-methyItransferase (EC 2.1.1) (involved i n the b i o s y n t h e s i s of epinephrine) has been s t u d i e d r e c e n t l y and found to methylate non-aromatic substrates as w e l l as aromatic ones (e.g. Figure 12, R = p r o p y l , c y c l o h e x y l , c y c l o h e x - 3 - e n y l , c y c l o - o c t y l ) . The enzyme has an absolute r e q u i r e ment f o r a hydroxy1 group at the 3 - p o s i t i o n on an e t h y l s i d e chain (72). Methylation r e a c t i o n s have not featured widely i n p e s t i c i d e metabolism; however, there i s enough information d e r i v e d from endogenous biochemistry and drug metabolism to be u s e f u l should 1

CRAWFORD AND i i u T S O N

Type

II

Metabolism

K3

->HS

Et

EtO

Ο

OH

Me S

MeS

mercapturic acid

Pr-N^^O

Glutathione in the metabolism of thiocarbamate herbicides

and thiotriazine

220


b i o t r a n s f o r m a t i o n without being obvious i n the f i n a l products. Even with the well-known glutathione-dependent demethylation of dimethylphosphate t r i e s t e r i n s e c t i c i d e s , most of the S-methylg l u t a t h i o n e formed i s metabolised to CO2 and very l i t t l e me t h y 1 mercapturic a c i d i s excreted. C l e a r l y the use of s u b c e l l u l a r f r a c t i o n s i s i n d i c a t e d i n d i s c o v e r i e s of t h i s type. The f a t e of the v i n y l phosphate i n s e c t i c i d e , dimethylvinphos, may be used to i l l u s t r a t e t h i s p o i n t f u r t h e r . The main r a d i o a c t i v e metabolites d e r i v e d from l^C-phenyl l a b e l l i n g i n v i v o were de-methyldimethylvinphos and a metabolite derived from the phenylvinyloxy group, 1-(2,4-dichlorophenyl)ethanol (as glucuronide) (4). The demethylation, p r e d i c t a b l y , was e f f e c t e d by ( d i a l y s e d ) c y t o s o l and g l u t a t h i o n e . 2,4-Dichlorophenylethanol was assumed to be d e r i v e d from 2,4-dichloroacetophenone which i n turn was thought to be formed by the r e d u c t i v e d e c h l o r i n a t i o n of the h y d r o l y s i s product of dimethylvinphos (2,4-dichlorophenacyl c h l o r i d e ) . The mechanism of the r e d u c t i v e d e c h l o r i n a t i o n was unknown u n t i l we s t u d i e d the metabolism of the phenacyl c h l o r i d e i n r a t l i v e r f r a c t i o n s . The f o l l o w i n g sequence of reactions was d i s c o v e r e d u s i n g c y t o s o l : ( i ) spontaneous r e a c t i o n of phenacyl h a l i d e with g l u t a t h i o n e to a f f o r d 2 , 4 - d i c h l o r o p h e n a c y l - g l u t a t h i o n e , ( i i ) enzyme-catalysed r e a c t i o n of the l a t t e r with another molecule of g l u t a t h i o n e to form o x i d i s e d g l u t a t h i o n e and the phenacyl anion which rearranged to 2,4-dichloroacetophenone (133). Microsomes and NADPH simply reduced the keto group of 2,4-dichlorophenacyl c h l o r i d e to give the c h l o r o h y d r i n (not observed i n the i n v i v o metabolism) . Thus g l u t a t h i o n e enters the metabolic pathways of dimethylvinphos at three p o i n t s (Figure 18) but i t i s s c a r c e l y observed i n the excreted m e t a b o l i t e s . In t h i s s e r i e s of experiments we a l s o used the i n v i t r o technique to demonstrate that the a d m i n i s t r a t i o n of phénobarbital to r a t s (which p r o t e c t e d them 1 0 - f o l d against the acute o r a l t o x i c i t y of dimethylvinphos) induced the a c t i v i t y of the c y t o s o l demethylating enzyme 2 - f o l d . Dimethylvinphos e x h i b i t s a large d i f f e r e n c e i n acute t o x i c i t y to r a t and dog ( r a t > dog); dog l i v e r c y t o s o l was shown to demethylate dimethylvinphos at about twice the rate of r a t l i v e r c y t o s o l (4). Thus, the enzyme could have some r o l e i n the s e l e c t i v e t o x i c i t y of dimethylvinphos. This example i l l u s t r a t e s s e v e r a l important f e a t u r e s : d i r e c t g l u t a t h i o n e conjugation of parent molecule ( e f f e c t i n g detoxification) ( i i ) g l u t a t h i o n e conjugation a f t e r b i o a c t i v a t i o n by primary metabolism ( h y d r o l y s i s i n t h i s case) ( i i i ) g l u t a t h i o n e conjugation by attack on an e l e c t r o p h i l i c atom other than carbon (sulphur i n t h i s case) ( i v ) only t r a c e s of the glutathione-conjugated products (mercapturic a c i d s ) appearing i n the excreted metabolites (v) use of s u b c e l l u l a r f r a c t i o n s to i n v e s t i g a t e a species d i f f e r e n c e ( r a t versus dog) (i)

6.

CRAWFORD AND HUTSON

GSH

II

Metabolism

GSH

CI Figure 18.

Type

221

GSH

CI

Glutathione in the metabolism of dimethylvinphos

222

XENOBIOTIC M E T A B O L I S M

( v i ) use of s u b c e l l u l a r f r a c t i o n s to i n v e s t i g a t e the of drug-metabolising enzymes of an animal.

alteration

Studies of the l i v e r enzyme have shown that g l u t a t h i o n e t r a n s f e r a s e s occur i n many other animals i n c l u d i n g r a b b i t (118), p i g (118), (23), monkey (23)(134)(135), t r e e shrew (23), sheep (136), guinea-pig (122), horse (122), cow (122), mouse (122), chicken (126), and man (137). Where d e t a i l e d s t u d i e s have been c a r r i e d out (135)(136) (137) the enzymes, i n c l u d i n g those of man, have been shown to be very s i m i l a r i n p h y s i c a l p r o p e r t i e s . L i v e r i s the r i c h e s t source of the enzymes. For example, a recent study of Japanese monkey (Macaca fuscata) (134) was t y p i c a l i n showing the f o l l o w i n g a r y l t r a n s f e r a s e ( 1 , 2 - d i c h l o r o 4-nitrobenzene) a c t i v i t i e s (μιιιοΐ/min/mg p r o t e i n ) : l i v e r , 18; spleen, 2.6; kidney, 2.1; lung, 1.9; b r a i n , 1.9; muscle, 1.8; p l a c e n t a , 0.3; pancreas, 0; e r y t h r o c y t e s , 0; blood, 0. A c t i v i t y has r e c e n t l y been reported i n small i n t e s t i n e s (138)(139) and i n leucocytes (140). Enzyme measurements have a l s o been used to assess glutathione conjugation during development of the neonate. A l k y l t r a n s f e r a s e a c t i v i t y i n the r a t neonate i s very low f o r about 6 days and then r i s e s s t e a d i l y f o r about 40 days to the adult l e v e l (141). Trans f e r a s e Β (measured as a r y l t r a n s f e r a s e ) i s present at b i r t h at about one f i f t h of the adult l e v e l and r i s e s s t e a d i l y f o r about 40 days (142). However, i n a l l general statements about the presence of g l u t a t h i o n e t r a n s f e r a s e i n various s p e c i e s , t i s s u e s or a l t e r e d s t a t e s , the s u b s t r a t e used f o r assay must be noted before the relevance to one's own work i s assessed. The importance of g l u t a t h i o n e conjugation i n l i m i t i n g the c y t o t o x i c , mutagenic and c a r c i n o g e n i c a c t i o n of e l e c t r o p h i l i c compounds (143) i s gener a t i n g much research i n t o the species and t i s s u e d i s t r i b u t i o n of the enzyme(s). However, too much r e l i a n c e on r e s u l t s d e r i v e d from t e s t s using substrates (e.g. p o l y c y c l i c aromatic hydrocarbon epoxides) unrelated to one's own problem may w e l l prove to be only of l i m i t e d v a l u e . T o x i c o l o g i c a l s i g n i f i c a n c e . Glutathione conjugation r e s u l t s i n a dramatic change i n the p h y s i c a l p r o p e r t i e s of a molecule, u s u a l l y l e a d i n g to a l o s s of b i o a c t i v i t y . The conjugate i s i d e a l l y s t r u c t u r e d f o r b i l i a r y s e c r e t i o n and t h e r e f o r e i t i s e f f i c i e n t l y removed from the l i v e r . Other enzymes e f f i c i e n t l y convert the conjugate i n t o a mercapturic a c i d that i s r e a d i l y excreted v i a the u r i n e . However, perhaps the most important f u n c t i o n of t h i s conjugation process i s the p r o t e c t i o n i t a f f o r d s against e l e c t r o p h i l i c compounds, be they ingested as such or generated w i t h i n the organism v i a metabolism. Without t h i s p r o t e c t i o n mammals would be much more s u s c e p t i b l e than they are to low doses of teratogens, mutagens, carcinogens and c y t o t o x i c compounds.

6.

CRAWFORD AND HUTSON

Type

II

Metabolism

223

Other Uses of S u b c e l l u l a r F r a c t i o n s i n X e n o b i o t i c Metabolism Studies A r e l a t i v e l y recent use of s u b c e l l u l a r f r a c t i o n s i s i n b a c t e r i a l t e s t systems f o r mutagenicity of the type developed by Ames and coworkers (144). The f r a c t i o n commonly used i s a 9000 g f r a c t i o n (S-9 f r a c t i o n ) from the l i v e r s of r a t s t r e a t e d with an A r a c h l o r (to induce microsomal enzymes). I t s r o l e i n the t e s t system i s the p r o v i s i o n of a mammalian metabolism c a p a b i l i t y f o r the ( p o s s i b l e ) a c t i v a t i o n of i n t r i n s i c a l l y i n a c t i v e compounds. This may not commonly be seen as a use i n metabolism s t u d i e s ; but i f the b a c t e r i a are regarded as a bioassay technique f o r the d e t e c t i o n of mutagens, the system i s a u s e f u l a d d i t i o n to the techniques used f o r the study of metabolism i n v i t r o . The main component of the b i o a c t i v a t i o n system i s regarded as being the microsomal mono-oxygenase (hence the use of inducers to prepare a more 'potent S-9 f r a c t i o n ) . However, i t w i l l be c l e a r from the v a r i o u s r e a c t i o n s discussed above that some of the type I I r e a c t i o n s e f f e c t the b i o a c t i v a t i o n of c e r t a i n m e t a b o l i t e s . They a l s o e f f e c t the d e a c t i v a t i o n of many compounds and t h e i r metabo l i t e s . The p o t e n t i a l l y great p r e d i c t i v e value of these t e s t systems has l e d to t h e i r widespread, but o f t e n u n c r i t i c a l use. The importance of a standardised p r e p a r a t i o n c o n t a i n i n g a c t i v e microsomal mono-oxygenase i s appreciated but the r o l e of the many other enzymes i n the S-9 f r a c t i o n has been l a r g e l y ignored. 1

The widespread use and e n t h u s i a s t i c r e c e p t i o n gained by t h i s simple, quick and p o t e n t i a l l y very u s e f u l system has l e d to a r e a c t i o n from c e r t a i n quarters and a heated debate i s c u r r e n t l y being conducted. There i s c l e a r l y a need to d e f i n e the r e a c t i o n s o c c u r r i n g i n the t e s t system. The balance between a c t i v a t i o n and d e a c t i v a t i o n i s c r i t i c a l to i t s relevance to the i n v i v o s i t u a t i o n . The s t a t e of the v a r i o u s enzymes i n the S-9 f r a c t i o n and the concentrations of the various c o f a c t o r s (many of which are described above) r e q u i r e s measurement and c o n t r o l . Species v a r i a t i o n s are important. For example i t i s p o s s i b l e that 2-aminoanthracene (mutagenic i n the presence of S-9 from r a t l i v e r ) would not give a p o s i t i v e response i f dog l i v e r S-9 f r a c t i o n s were used. The f i r s t step i n i t s b i o a c t i v a t i o n ( N - a c e t y l a t i o n , see Figures 9 and 10) i s i n o p e r a t i v e . Glutathione-dependent d e a c t i v a t i o n i s l a r g e l y i n o p e r a t i v e i n standard S-9 f r a c t i o n because, although the g l u t a t h i o n e S-transferases are present, g l u t a t h i o n e i t s e l f i s l a r g e l y destroyed by catabolism, d i l u t i o n and o x i d a t i o n (145). A study of the various enzyme a c t i v i t i e s and c o f a c t o r concentrations i n human l i v e r f r a c t i o n (prepared as S-9) would a l s o prove very u s e f u l i n the i n t e r p r e t a t i o n of r e s u l t s of the Ames t e s t .


224

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15. 16. 17. 18. 19. 20. 21. 22. 23.

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24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51.

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75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101.

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