Techniques for Studying the Metabolism of Xenobiotics by Intact

4 Techniques for Studying the Metabolism of Xenobiotics by Intact Animal Cells, Tissues, and Organs In Vitro

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 11, 2016 | http://pubs.acs.org Publication Date: April 5, 1979 | doi: 10.1021/bk-1979-0097.ch004

ROBERT E. MENZER Department of Entomology, University of Maryland, College Park, M D 20742

Researchers studying t h e f a t e o f x e n o b i o t i c s i n mammals have tended t o concentrate t h e i r e f f o r t s e i t h e r on the whole animal o r on l i v e r homogenate systems such as microsomes. Each, o f course, has i t s advantages. The response o f a whole animal most c l o s e l y represents what i s l i k e l y t o occur upon exposure o f man t o a compound. L i v e r homogenates are u s e f u l i n that the l i v e r i s t h e p r i n c i p a l organ r e s p o n s i b l e f o r the degradation and e l i m i n a t i o n of x e n o b i o t i c s from mammalian systems, and l i v e r microsomes appear t o be the p r i n c i p a l s i t e w i t h i n the mammal where metabolic conversions take p l a c e . These systems a l l o w a r a p i d , inexpensive e v a l u a t i o n o f metabolic events which a r e l i k e l y to take p l a c e i n the mammal f o r any x e n o b i o t i c . The combination o f s t u d i e s i n whole animals and l i v e r microsomal systems g e n e r a l l y provides a good understanding o f the f a t e o f a x e n o b i o t i c i n a mammalian system. O c c a s i o n a l l y , however, a compound w i l l be i n v e s t i g a t e d i n which the events t a k i n g p l a c e i n a l i v e r microsomal system do not e n t i r e l y m i r r o r what occurs i n the whole organism. Furthermore, the use o f microsomal systems does not p r o v i d e i n f o r m a t i o n on t h e pharmacodynamics i n v o l v e d i n t h e a b s o r p t i o n , d i s t r i b u t i o n , and e l i m i n a t i o n o f a x e n o b i o t i c from an organism. Other techniques are a v a i l a b l e which w i l l p r o v i d e a d d i t i o n a l i n f o r m a t i o n on t h e f a t e o f a x e n o b i o t i c i n a mammal that would be d i f f i c u l t to o b t a i n e i t h e r w i t h l i v e r microsomal systems o r i n whole organisms. In t h i s paper we w i l l examine the use o f perfused organ systems, w i t h p a r t i c u l a r emphasis on l i v e r and lung p e r f u s i o n ; t i s s u e s l i c e s , p a r t i c u l a r l y from lung and l i v e r ; c e l l c u l t u r e systems, both primary c e l l c u l t u r e s and e s t a b l i s h e d c e l l l i n e s ; and f i n a l l y , the promising new technique o f i s o l a t e d hepatocyte preparat i o n s . For each technique we w i l l examine the methodology c u r r e n t l y i n use and evaluate the ease by which an i n v e s t i g a t o r inexperienced i n the use o f t h e technique would be a b l e t o adopt i t f o r s t u d i e s on s p e c i f i c compounds. The a p p l i c a t i o n o f each o f these techniques t o the study o f x e n o b i o t i c s , w i t h p a r t i c u l a r emphasis on p e s t i c i d e s , w i l l be i l l u s t r a t e d . 0-8412-0486-l/79/47-097-131$05.00/0 © 1979 American Chemical Society

Paulson et al.; Xenobiotic Metabolism: In Vitro Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Organ P e r f u s i o n As i s the case with most in vitro mammalian p r e p a r a t i o n s , organ p e r f u s i o n was o r i g i n a l l y developed to study the physiology and biochemistry o f the organ i t s e l f and the f u n c t i o n a l p o s i t i o n o f the organ i n the metabolism o f the normal animal. Organ perf u s i o n s t u d i e s are an intermediate step between s t u d i e s i n the whole animal and experiments with i s o l a t e d s u b c e l l u l a r preparations. By observing the response o f a v i a b l e organ i s o l a t e d from the system i n which i t r e s i d e s , one i s a b l e to assess the p a r t which the organ p l a y s i n the metabolism o f any given system. I t i s f r e q u e n t l y p o s s i b l e to observe s p e c i f i c steps i n metabolic processes i n an i s o l a t e d organ when i n the whole animal only subs t r a t e and f i n a l product are observable. I t i s i n the a b i l i t y o f the r e s e a r c h e r to i s o l a t e i n d i v i d u a l r e a c t i o n s that perfused organs have t h e i r p r i n c i p a l u t i l i t y . Another advantage o f perfused organ p r e p a r a t i o n s i s that blood flow, gas exchange, and temperature are under d i r e c t c o n t r o l . T h i s enables the r e s e a r c h e r to d e l i b e r a t e l y manipulate these parameters to assess the e f f e c t of, f o r example, reduced blood flow, anoxia, hypothermia, changes i n blood pH, or o s m o l a r i t y on the metabolic process being s t u d i e d . For background i n f o r m a t i o n and d e s c r i p t i o n s o f the methodology a v a i l a b l e f o r organ p e r f u s i o n one may r e f e r to a number o f books d e a l i n g with the s u b j e c t . Two examples are R i t c h i e and Hardc a s t l e (11) and Ross (12). P e r f u s i o n may be d e f i n e d as the passage of a f l u i d medium or blood through the v a s c u l a r bed o f an organ. I t i s easy to see how one can u t i l i z e a p e r f u s i n g organ to study the metabolism o f a x e n o b i o t i c by the simple i n t r o d u c t i o n o f the chemical i n t o the p e r f u s a t e and o b s e r v a t i o n o f the e f f e c t which the organ has on the chemical. The p o s s i b i l i t i e s f o r continuous sampling of the p e r f u s a t e , continuous a d d i t i o n or p u l s e a d d i t i o n o f the chemical to the p e r f u s a t e , and s t u d i e s of the i n t e r a c t i o n s o f more than one chemical i n organs are r e a d i l y apparent. For workers studying x e n o b i o t i c s the two most important organ p e r f u s i o n systems are those f o r the l i v e r and the lungs. The l i v e r i s probably the most f r e q u e n t l y perfused organ and i s used f o r a wide v a r i e t y o f s t u d i e s . The techniques f o r l i v e r p e r f u s i o n p r e s e n t l y most f r e q u e n t l y used are those developed by M i l l e r et al. (7) as i l l u s t r a t e d i n F i g u r e 1. The apparatus designed by M i l l e r and co-workers i s now commercially a v a i l a b l e . Techniques f o r p e r f u s i o n o f the lung have been more r e c e n t l y developed, the system o f Niemeier and Bingham (8) being most f r e quently r e f e r r e d to by workers i n t h i s area (9). Niemeier and Bingham developed a system f o r the p e r f u s i o n of r a b b i t lungs which allows the use o f u n d i l u t e d autologous whole blood as the p e r f u sate. Lung p e r f u s i o n i s more complicated than l i v e r s i n c e one i s able to p r o v i d e both a c i r c u l a t i n g l i q u i d p e r f u s a t e as w e l l as being able to v e n t i l a t e the i s o l a t e d lung with a gas system. In


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Figure 1. Liver perfusion apparatus. The apparatus is enclosed in a temperatureregulated cabinet and is composed of a system for pumping the perfusate (blood) at constant hydrostatic pressure and a system for oxygenating the blood (7, 12).



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a lung, t h e r e f o r e , one can introduce a x e n o b i o t i c to the system e i t h e r through the p e r f u s i o n system i t s e l f or through the gas v e n t i l a t i n g system, thus approximating the i n t r o d u c t i o n o f the x e n o b i o t i c e i t h e r through the c i r c u l a t o r y system or the r e s p i r a t o r y system i n the whole animal. The use o f lung p e r f u s i o n i s p a r t i c u l a r l y advantageous because the e s t i m a t i o n o f s u b s t r a t e u t i l i z a t i o n by the lung i s very d i f f i c u l t in vivo. Lung t i s s u e s l i c e s or other in vitro p r e p a r a t i o n s do not seem to approximate the p h y s i o l o g i c a l s t a t e o f the whole organ as w e l l as comparable preparations from the l i v e r ; the need f o r oxygen and the problem o f d i f f u s i o n are p a r t i c u l a r problems i n the lung. I s o l a t e d perfused lung systems overcome these problems and w i l l be p a r t i c u l a r l y u s e f u l i n the f u t u r e f o r the study o f the metabolism by the lung o f x e n o b i o t i c s introduced by i n h a l a t i o n . X e n o b i o t i c metabolism i n perfused l i v e r may be i l l u s t r a t e d with p a r a t h i o n . Both p a r a t h i o n and paraoxon were s t u d i e d to assess the metabolic r e l a t i o n s h i p s o f these compounds i n the l i v e r ( 3 ) . I t was shown that 68% o f the administered p a r a t h i o n was metabolized to water s o l u b l e compounds. These water s o l u b l e compounds were found to be conjugates o f p - n i t r o p h e n o l , the bulk o f which was i n the c i r c u l a t i n g p e r f u s a t e , not a s s o c i a t e d with the l i v e r t i s s u e or excreted v i a the b i l e . An a d d i t i o n a l 2.5% was paraoxon and there were traces o f unconjugated p - n i t r o p h e n o l . Administered paraoxon was degraded almost e n t i r e l y (98.5%) to water s o l u b l e compounds, which again were conjugates o f p - n i t r o phenol . Another type of experiment p o s s i b l e using perfused organ systems i s i l l u s t r a t e d by a study o f mirex-induced suppression o f b i l i a r y e x c r e t i o n o f p o l y c h l o r i n a t e d biphenyls ( 5 ) . In t h i s study i t was shown t h a t 50 mg/kg/day o f mirex-pretreatment o f the r a t s whose l i v e r s were perfused suppressed the b i l i a r y e x c r e t i o n o f 4-chlorobiphenyl and i t s metabolites by 92%. Furthermore, the r a t e o f metabolism o f 4-chlorobiphenyl was decreased s l i g h t l y by mirex pretreatment. The reason f o r t h i s phenomenon was t h e o r i z e d to be that t r a n s p o r t o f otherwise r e a d i l y e x c r e t a b l e metabolites from the hepatocytes i n t o the b i l e c a n a l i c u l i was a f f e c t e d by mirex. The f a c t that mirex causes changes i n the a b i l i t y o f the l i v e r to excrete x e n o b i o t i c s has i m p l i c a t i o n s f o r the p o s s i b l e e f f e c t o f t h i s compound on the t o x i c o l o g y o f other compounds. A number o f recent s t u d i e s o f x e n o b i o t i c s i n perfused lung systems have been reported: a l d r i n and d i e l d r i n (6), p a r a t h i o n , methadone, imipramine, c h l o r c y c l i z i n e , and p e n t o b a r b i t a l ( 4 ) , t r i c h l o r o e t h y l e n e (2), and c a r b a r y l (1). These s t u d i e s i l l u s t r a t e w e l l the p o t e n t i a l f o r important r e s u l t s which can be obtained from organ p e r f u s i o n s t u d i e s . Perfused r a b b i t lungs were used t o study the metabolism and b i n d i n g p r o p e r t i e s o f a l d r i n and d i e l d r i n . The compounds were added to the system v i a the p e r f u s i o n medium and samples were withdrawn at s e v e r a l i n t e r v a l s . I t was noted that a l d r i n was epoxidized t o d i e l d r i n , but d i e l d r i n was not f u r t h e r metabolized



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i n the system; no epoxide hydrase a c t i v i t y could be detected. The uptake of a l d r i n and d i e l d r i n was by d i f f u s i o n . The r a t e of uptake was b i p h a s i c , c o n s i s t i n g of an i n i t i a l r a p i d phase f o l l o w ed by a slower one, r e l a t e d to conversion o f a l d r i n to d i e l d r i n i n the case of a l d r i n . These s t u d i e s show that the lungs are not a s i g n i f i c a n t storage s i t e f o r e i t h e r compound (6). The metabolism o f p a r a t h i o n , methadone, imipramine, c h l o r c y c l i z i n e , and p e n t o b a r b i t a l was compared i n a r a b b i t perfused lung p r e p a r a t i o n with r a b b i t lung and l i v e r microsomal preparations. In the perfused lung p a r a t h i o n , methadone, and pentob a r b i t a l were o x i d a t i v e l y metabolized. Parathion was e x t e n s i v e l y metabolized to paraoxon and water s o l u b l e m e t a b o l i t e s , which were not f u r t h e r i d e n t i f i e d . No accumulation of p a r a t h i o n was observed i n the system. With i n c r e a s i n g p e r f u s i o n time there was a decrease i n the appearance o f p a r a t h i o n , methadone, and pentob a r b i t a l m e t a b o l i t e s . T h i s was more l i k e l y a s s o c i a t e d with decreasing s u b s t r a t e or d e p l e t i o n o f c o f a c t o r than to denatura t i o n or d e s t r u c t i o n o f the lung system. No s i g n i f i c a n t d i f f e r ences were observed between the drug m e t a b o l i z i n g a c t i v i t i e s o f the microsomes o f lung or l i v e r and the perfused lung system ( 4 ) . An i n t e r e s t i n g example o f the use o f the perfused lung system to study the metabolism o f a x e n o b i o t i c by the i n h a l a t i o n route i s given by Dalbey and Bingham (2). They s t u d i e d the metabolism of t r i c h l o r o e t h y l e n e i n a r a b b i t perfused lung system. T r i c h l o r o e t h y l e n e was generated i n t o the a i r s u p p l i e d to the i s o l a t e d perfused lungs, and the compound and i t s metabolites were measured p e r i o d i c a l l y i n the p e r f u s a t e and i n the lung t i s s u e f o l l o w i n g a three-hour p e r f u s i o n p e r i o d . T r i c h l o r o e t h y l e n e was e x t e n s i v e l y metabolized to t r i c h l o r o e t h a n o l , t r i c h l o r o e t h a n o l glucuronide, and t r i c h l o r o a c e t i c a c i d . I t was p o s t u l a t e d that c h l o r a l hydrate was an intermediate i n the metabolism o f t r i chloroethylene but i t was not i s o l a t e d i n t h i s system. C a r b a r y l metabolism i n the perfused r a b b i t lung was shown to be r a p i d . The pharmacokinetics of c a r b a r y l uptake demonstrated simple d i f f u s i o n . A f t e r 30 minutes o f p e r f u s i o n 1-naphthol was seen i n the p e r f u s a t e e x t r a c t s . Since i t s c o n c e n t r a t i o n i n the p e r f u s a t e decreased during the course o f the experiment, i t was concluded that the 1-naphthol which was taken up by the lungs was formed by nonenzymatic h y d r o l y s i s of c a r b a r y l i n the p e r f u s a t e . 4-Hydroxycarbaryl appeared i n the p e r f u s a t e at 30 minutes and i n creased i n c o n c e n t r a t i o n u n t i l 60 minutes, a f t e r which i t decreased. Other metabolites were i s o l a t e d but no attempt was made to i d e n t i f y them ( 1 ) . Comparative s t u d i e s (10) have shown that perfused organs, e s p e c i a l l y the l i v e r , p a r a l l e l changes which occur i n the whole organism. Thus, the technique can be a u s e f u l b r i d g e between other in vitro s t u d i e s and in vivo s t u d i e s .


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The techniques f o r t i s s u e s l i c e s t u d i e s o f metabolism were probably f i r s t introduced by Otto Warburg i n 1923 (19). The method caught on r a p i d l y and was used by many workers so extens i v e l y that Krebs and H e n s e l e i t , w r i t i n g i n 1932 (15), noted that between the two methods a v a i l a b l e f o r studying metabolism i n animal t i s s u e s , they p r e f e r r e d t i s s u e s l i c e s over p e r f u s i o n . T h i s judgment probably represented a r e a c t i o n to the lack o f r e p r o d u c i b i l i t y o f p e r f u s i o n techniques and the complicated systems then i n use. By comparison, t i s s u e s l i c e s coupled with manometry o f f e r e d a simple, p r o d u c i b l e , f l e x i b l e method f o r studying metabolism. While the judgment made by Krebs and H e n s e l e i t would probably not be v a l i d today, the i n t e r v e n i n g years have seen a great deal o f f i n e work using t h e i s o l a t e d t i s s u e s l i c e technique. Again, the l i v e r i s the p r i n c i p a l organ which has been s t u d i e d using t i s s u e s l i c e s . However, many other organs have a l s o been used i n a v a r i e t y o f s t u d i e s . Of p a r t i c u l a r note f o r x e n o b i o t i c metabolism s t u d i e s i n a d d i t i o n to the l i v e r a r e kidneys, lungs, and i n t e s t i n e s . The key to the p r e p a r a t i o n o f v i a b l e t i s s u e s l i c e s i s to o b t a i n r e p r o d u c i b l e , t h i n s l i c e s , g e n e r a l l y l e s s than 0.5 mm t h i c k . T h i s i s most f r e q u e n t l y done at present using a microtome or s i m i l a r instrument. The v i a b i l i t y o f the t i s s u e and s t a n d a r d i z a t i o n to determine r e p r o d u c i b i l i t y are f r e q u e n t l y evaluated using Warburg respirometry (16). Thin s l i c e techniques have been used e x t e n s i v e l y f o r the study o f p e s t i c i d e metabolism, and examples o f a v a r i e t y o f s t u d i e s f o l l o w . One o f the e a r l i e s t establishments o f the need f o r conversion of an organophosphorus i n s e c t i c i d e t o an a c t i v e a n t i c h o l i n e s t e r a s e metabolite used the l i v e r s l i c e technique i n combination with Warburg respirometry (14) . L i v e r s l i c e s were used t o demonstrate the conversion o f dimefox, a phosphoramidate, to an a c t i v e i n h i b i t o r using r a t b r a i n c h o l i n e s t e r a s e as the substrate. I n h i b i t i o n o f c h o l i n e s t e r a s e was measured i n the Warburg apparatus, and i n h i b i t i o n o f c h o l i n e s t e r a s e was taken as i n d i r e c t evidence f o r metabolism o f dimefox by the l i v e r s l i c e . Liver s l i c e s 0.5 mm t h i c k and 5 mm square, washed twice with 0.9% sodium c h l o r i d e , were used i n each Warburg f l a s k . Substrate and enzyme sources were added from s i d e arms, and c h o l i n e s t e r a s e i n h i b i t i o n was assayed by standard methodology. The h e r b i c i d e s propham and chloropropham were s t u d i e d i n the r a t in vivo and metabolism was compared i n l i v e r s l i c e s and kidney s l i c e s (13) . These h e r b i c i d e s were metabolized in vivo to two major and three minor metabolites; both o x i d a t i v e and h y d r o l y t i c mechanisms were evident. L i v e r and kidney s l i c e s , however, d i d not hydrolyze the c h a i n moiety as observed in vitro. Only l i v e r s l i c e s converted the h e r b i c i d e s to t h e i r o x i d a t i v e m e t a b o l i t e s . Rat r e n a l c o r t i c a l s l i c e s and r a t l i v e r s l i c e s were used t o assess the mechanism f o r e x c r e t i o n o f d i c h l o r o d i p h e n y l a c e t i c a c i d (DDA) from animals (18) . I t was hypothesized that DDA i s excreted



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v i a the organic a c i d system. I t was shown that DDA was a v i d l y accumulated by l i v e r and by kidney s l i c e s . DDT o r organic bases d i d not i n h i b i t the accumulation of DDA, i n d i c a t i n g that the organic a c i d e x c r e t i o n system i s indeed r e s p o n s i b l e f o r the e l i m i n a t i o n o f DDA from the mammalian system. This u l t i m a t e l y , o f course, i s the method f o r the e l i m i n a t i o n o f DDT from the system s i n c e DDT i s f i r s t converted t o DDA before i t i s excreted. An i n t e r e s t i n g study o f c a r b a r y l metabolism has been reported using an i s o l a t e d i n t e s t i n a l t i s s u e (17) . For t h i s study the small i n t e s t i n e between the b i l e duct and the cecum was removed from male r a t s , r i n s e d i n i s o t o n i c s a l i n e , and d i v i d e d i n t o three approximately equal p a r t s . The s e c t i o n s were everted and maint a i n e d over i c e i n f r e s h s a l i n e plus glucose s o l u t i o n u n t i l the s e r o s a l compartment was f i l l e d with s e r o s a l f l u i d . The f i l l e d sacs were then t r a n s f e r r e d to a f l a s k c o n t a i n i n g mucosal f l u i d and incubated with a g i t a t i o n f o r one or two hours. Analysis of the products o f c a r b a r y l i n c u b a t i o n i n d i c a t e d the p r o d u c t i o n o f 1-naphthol, again apparently by nonenzymatic mechanisms. At l e a s t seven metabolites were i d e n t i f i e d . The p r i n c i p a l water s o l u b l e metabolite (60%) was 1-naphthol glucuronide. Although much u s e f u l i n f o r m a t i o n about x e n o b i o t i c metabolism has been obtained with l i v e r s l i c e techniques, most workers today p r e f e r to use other methods, probably because of d i f f i c u l t i e s o f r e p r o d u c i b i l i t y u s i n g the technique. C e l l Culture Mammalian c e l l s i n c u l t u r e have been used f o r over one-half century to study v a r i o u s aspects of b i o l o g y . Since H a r r i s o n f i r s t s u c c e s s f u l l y propagated medullary t i s s u e in vitro i n 1907, c e l l c u l t u r e s have been u t i l i z e d i n the study o f r a d i o b i o l o g y , c e l l d i v i s i o n , and g e n e t i c cytology as w e l l as other areas o f c e l l b i ology. Mammalian c e l l c u l t u r e s have a l s o been used to study the c o r r e l a t i o n o f c y t o t o x i c i t y of drugs with other pharmacological a t t r i b u t e s . The primary use o f c e l l c u l t u r e s has been to p r o v i d e a method f o r i n v e s t i g a t i n g the d i r e c t a c t i o n o f drugs and other chemicals on c e l l s i n the absence o f the complex i n t e r a c t i o n s which apply i n the whole animal. A high c o r r e l a t i o n , f o r example, has been found between in vitro c y t o t o x i c i t y and in vivo antitumor a c t i v i t y o f a number of chemotherapeutic agents screened f o r a n t i cancer a c t i v i t y . A wide v a r i e t y o f c e l l types have been c u l t u r e d and maintained in vitro. Both normal c e l l s from many d i f f e r e n t organs and t i s s u e s of many d i f f e r e n t animal species and abnormal c e l l s i s o l a t e d from tumors or other abnormal t i s s u e s have been u t i l i z e d . C e l l s have been s u c c e s s f u l l y explanted from animals i n a l l stages of development, from the embryo to the a d u l t . One of the problems which must be r e c o g n i z e d i n working with c e l l c u l t u r e s i s the d i f f e r e n c e which i s l i k e l y to e x i s t between these c e l l s t r a i n s and the t i s s u e from which they o r i g i n a t e d .



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C e l l s t r a i n s or c e l l l i n e s , so c a l l e d , c o n s i s t o f c e l l s which may have been growing in vitro f o r a c o n s i d e r a b l e length o f time and which have undergone many subcultures or d i l u t i o n s o f c e l l numbers. G e n e r a l l y they have a d i f f e r e n t p a t t e r n o f metabolism, are capable o f supporting the growth o f a wide v a r i e t y of v i r u s e s and microorganisms, and are f r e q u e n t l y p o l y p l o i d . Such d i v e r gences from normal t i s s u e must be considered when e v a l u a t i n g experimental r e s u l t s . Some o f the d i f f i c u l t i e s i n working with c e l l l i n e s are e l i m i n a t e d by working i n primary c e l l c u l t u r e s . These are c e l l s which have not undergone even a s i n g l e passage or subculture s i n c e having been explanted from the donor animal. Such c u l t u r e s have been shown to r e t a i n enzymatic a c t i v i t i e s s i m i l a r to those of the in vivo donor t i s s u e . The a c t i v i t y u s u a l l y l a s t s through a few i n i t i a l subcultures before receding to decreased l e v e l s . C e l l s o f both types, c e l l l i n e s and primary c e l l c u l t u r e s , are commercially a v a i l a b l e . The techniques f o r maintaining and using c e l l s are simple, although r i g o r o u s s t e r i l e technique i s a b s o l u t e l y necessary t o avoid contamination o f c e l l c u l t u r e s by invading microorganisms o f a l l types. The media used f o r c e l l c u l t u r e s are i d e a l f o r the growth of microorganisms, which, i f they contaminate the c u l t u r e s , lead to a r t i f a c t s i n the experimental r e s u l t s . Rigorous a t t e n t i o n to s t e r i l e technique i s a p r e r e q u i s i t e to accurate i n t e r p r e t a t i o n o f the r e s u l t s o f r e search u s i n g c e l l c u l t u r e s . Only a few papers appear i n the l i t e r a t u r e r e p o r t i n g the r e s u l t s of s t u d i e s o f p e s t i c i d e metabolism i n c u l t u r e d mammalian cells. The v a r i o u s s t u d i e s have used human embryonic lung L-132 c e l l s , HeLa S c e l l s , mouse f i b r o b l a s t L-929 c e l l s , and mouse L-5178 lymphoma c e l l s . A l l o f these are e s t a b l i s h e d c e l l l i n e s . In a d d i t i o n , s t u d i e s have been conducted using primary human embryonic lung c e l l s . .DDT metabolism has been s t u d i e d i n HeLa S c e l l s (22), mouse L-5178 lymphoma c e l l s (29), and primary human embryonic lung c e l l s (28). The v a r i a t i o n i n the s u s c e p t i b i l i t y o f a compound to metabolism by v a r i o u s c e l l types i s i l l u s t r a t e d i n these s t u d i e s . Mouse lymphoma c e l l s grown f o r 72 hours i n the presence o f DDT f a i l e d to metabolize the compound. At the other end of the spectrum, HeLa S c e l l s metabolized DDT to DDD, DDE, DBM, and DBP. DDE was the metabolite present i n the h i g h e s t q u a n t i t y a f t e r 24 hours o f i n c u b a t i o n and was p o s t u l a t e d t o be the t e r m i n a l metabo l i t e i n t h i s system. I t i s noted i n the paper, however, that the conversion o f DDT t o DDE could have been enhanced by the i r o n p o r p h y r i n complexes i n the medium. Primary human embryonic lung c e l l s metabolized DDT only to DDD (38%) and DDA ( 4 % ) . No other metabolites were found i n t h i s c e l l c u l t u r e system. C a r b a r y l has been s t u d i e d i n both a human embryonic lung c e l l l i n e , the L-132 s t r a i n (20, 21) and i n primary human embryonic lung c e l l s (23). The HEL c e l l l i n e was apparently a c t i v e i n conj u g a t i n g c a r b a r y l m e t a b o l i t e s . The water s o l u b l e aglycones r e -



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s u i t i n g from beta-glucuronidase and a r y l s u l f a t a s e treatment were 4- hydroxycarbaryl and 5,6-dihydro-5,6-dihydroxycarbaryl. In a d d i t i o n , h y d r o x y l a t i o n a t the C-4 p o s i t i o n i n c o n j u n c t i o n with h y d r o l y s i s of the carbamate group r e s u l t e d i n the formation o f naphthalene-1,4-diol. The i n t e r e s t i n g m e t a b o l i t e , 1-naphthyl methylcarbamate-tf-glucuronide, was a l s o r e p o r t e d i n t h i s study. The r e s u l t s obtained i n primary HEL c e l l s agreed v e r y c l o s e l y with those from the HEL c e l l l i n e . In the primary c e l l c u l t u r e s 1-naphthol was the p r i n c i p a l m e t a b o l i t e i s o l a t e d . Others i n c l u d ed n a p h t h a l e n e - l , 4 - d i o l , naphthalene-1,5-diol, 4-hydroxycarbary1, 5- hydroxycarbaryl, and 5,6-dihydro-5,6-dihydroxycarbaryl. In a d d i t i o n s i g n i f i c a n t amounts o f the administered c a r b a r y l were present i n e x t r a c t s as conjugates. A c i d h y d r o l y s i s f r e e d 4hydroxycarbary1, naphthalene-1,4-diol, and 5,6-dihydro-5,6-dihydroxycarbaryl. On the other hand, beta-glucuronidase treatment o f the aqueous m a t e r i a l d i d not f r e e aglycones. T h i s r e s u l t was i n agreement with the e a r l i e r work i n which Baron and Locke post u l a t e d the formation of an #-glucuronide i n the c e l l c u l t u r e system. The use o f both primary and e s t a b l i s h e d HEL c e l l s i s an important l i n k i n understanding the metabolism o f c a r b a r y l i n mammalian systems. The u t i l i t y and value o f c e l l c u l t u r e s t u d i e s i s i l l u s t r a t e d w e l l by a study o f dimethoate metabolism i n primary human embryo n i c lung c e l l s (27). In t h i s study the c e l l s were shown t o o x i d i z e dimethoate with no i n t e r f e r e n c e from competing h y d r o l y t i c r e a c t i o n s . Thus, the p r o g r e s s i o n o f dimethoate metabolism from the phosphorodithioate to the phosphorothioate, concomitant with o x i d a t i v e #-dimethylation o f both the phosphorodithioate and phosphorothioate, c o u l d be demonstrated (Figure 2 ) . The r e l a t i o n ships e x i s t i n g between these three metabolites and t h e i r parent compound could not be e s t a b l i s h e d so w e l l i n a system i n which h y d r o l y t i c r e a c t i o n were competing with the o x i d a t i v e ones. S t u d i e s o f the a c a r a c i d e chlorphenamidine (24), and the r e l a t e d phenylurea h e r b i c i d e s , c h l o r o t o l u r o n , fluometuron, and metobromuron, (25) revealed s t r i k i n g d i f f e r e n c e s i n the suscept i b i l i t y o f these m a t e r i a l s to metabolism by the c e l l s . On the one hand, chlorphenamidine was very s u s c e p t i b l e to o x i d a t i v e metabolism i n HEL c e l l s with the formation o f n e a r l y 82% o f the iV-formyl m e t a b o l i t e and 2% o f 4 - c h l o r o - o - t o l u i d i n e as w e l l as small q u a n t i t i e s o f other m e t a b o l i t e s . While the h e r b i c i d e s were very r e s i s t a n t t o metabolism ( l e s s than 2% o f the a p p l i e d compound was metabolized i n 72 hours i n each c a s e ) , the small q u a n t i t i e s of m e t a b o l i t e s that were formed were the r e s u l t o f o x i d a t i v e reactions i n c e l l s . I t was i n t h i s c e l l c u l t u r e system that the formation o f #-formyl d e r i v a t i v e s o f c h l o r o t o l u r o n was f i r s t observed, a r e s u l t l a t e r corroborated i n l i v e r microsomal prepa r a t i o n s and i n r a t s in vivo (26). The chromatographic behavior o f the formyl d e r i v a t i v e s made t h e i r d e t e c t i o n d i f f i c u l t i n systems which more a c t i v e l y metabolized the compound because o f i n t e r f e r i n g m a t e r i a l s on t h i n l a y e r chromatographic p l a t e s .


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I N C U B A T I O N Pesticide Biochemistry and Physiology

Figure 2. Dimethoate disappearance, organoextractable metabolite formation, and percentage of protein increase in HEL cell cultures incubated with C-dimethoate. Disappearance of dimethoate corresponds with the appearance of the des-N-methyl metabolite and the oxygen analog, which in turn disappears corresponding with the increase of its des-N-methyl derivative (27). 14


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C e l l c u l t u r e techniques are u s e f u l i n the study o f xenob i o t i c metabolism. They cannot be used t o e s t a b l i s h q u a n t i t a t i v e r e l a t i o n s h i p s between x e n o b i o t i c s and t h e i r m e t a b o l i t e s , p a r t i c u l a r l y when r e f e r e n c e to the whole animal i s d e s i r e d , but they can be i n v a l u a b l e i n studying the mechanisms o f metabolism and i n c h a r a c t e r i z i n g metabolites which may appear i n only minor q u a n t i t i e s i n other mammalian systems. Isolated

Hepatocytes

The p r e p a r a t i o n and use o f i s o l a t e d hepatocytes i s a r e c e n t i n n o v a t i o n i n the study o f x e n o b i o t i c metabolism and should prove to be most u s e f u l . The technique i s b a s i c a l l y a simple one. L i v e r c e l l s are d i s s o c i a t e d from the p r o t e i n matrix o f the organ and are i s o l a t e d i n v i a b l e c o n d i t i o n f o r use much as i f they were i s o l a t e d c e l l s i n c u l t u r e as d e s c r i b e d e a r l i e r . A v a r i e t y o f methods have been developed f o r the i s o l a t i o n of hepatocytes, although the techniques have not yet reached the r o u t i n e stage so that a l l r e s e a r c h e r s agree on the same b a s i c methodology f o r t h e i r p r e p a r a t i o n . E a r l i e r techniques i n v o l v e d e i t h e r ( l ) t h e perf u s i o n of l i v e r with calcium c h e l a t o r s or a l k a l i n e hyperosmolar s a l t s o l u t i o n s , (2) the d i g e s t i o n of l i v e r p i e c e s i n t e t r a p h e n y l boron, a potassium c h e l a t o r , (3) the use o f enzymes as d i s s o c i a t ing agents, n o t a b l y t r y p s i n f o r f e t a l o r neonatal m a t e r i a l , and (4) collagenase/hyaluronidase d i g e s t i o n . Various combinations o f these techniques have a l s o been t r i e d (40). C u r r e n t l y there appear t o be b a s i c a l l y two methods f o r the p r e p a r a t i o n o f hepatocytes i n general use. One i n v o l v e s the p e r f u s i o n of i s o l a t e d l i v e r with Hank's b u f f e r f o r approximately 15 minutes f o l l o w e d by the a d d i t i o n of collagenase to the p e r f u s a t e f o r an a d d i t i o n a l 10 to 15 minutes. T h i s treatment c o l l a p s e s the l i v e r , a f t e r which i t i s minced, c e n t r i f u g e d , washed, and resuspended i n Hank's b u f f e r f o r immediate use (45, 46, 47). The other technique e l i m inates the n e c e s s i t y f o r p e r f u s i o n and uses enzymatic d i s s o c i a t i o n by treatment o f l i v e r with c o l l a g e n a s e / h y a l u r o n i d a s e i n Hank's balanced s a l t s o l u t i o n f o l l o w e d by c e n t r i f u g a t i o n , washing, and resuspension (32, 40). The l a t t e r technique has the advantage o f s i m p l i c i t y and low cost s i n c e no s p e c i a l p e r f u s i o n apparatus i s needed. Furthermore, i t would be p o s s i b l e to prepare hepatocytes from p i e c e s of f r e s h l i v e r which might, f o r example, be a v a i l a b l e from b i o p s i e s without the need f o r the whole organ as r e q u i r e d i n the p e r f u s i o n technique. Both techniques appear to g i v e good y i e l d s o f v i a b l e c e l l s which can be used f o r metabo l i s m s t u d i e s . As f o r most in vitro methods, most o f the e a r l y developmental work on i s o l a t i o n o f v i a b l e hepatocytes has been done with r a t l i v e r s . However, there i s no reason why the t e c h niques developed cannot be a p p l i e d to the l i v e r s o f other s p e c i e s as w e l l , perhaps with very l i t t l e m o d i f i c a t i o n . In f a c t chicken hepatocytes have been i s o l a t e d using the r a t l i v e r techniques and were shown to c a r r y out normal biochemical f u n c t i o n s (37, 38).



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The c r i t e r i a o f v i a b i l i t y o f i s o l a t e d l i v e r c e l l s are a matter o f c o n s i d e r a b l e concern f o r metabolism s t u d i e s s i n c e r e p r o d u c i b i l i t y demands a technique f o r s t a n d a r d i z a t i o n o f prep a r a t i o n s made at d i f f e r e n t times or by d i f f e r e n t methods. The most u n i v e r s a l l y used technique to assess v i a b i l i t y o f i s o l a t e d hepatocytes i s the trypan blue e x c l u s i o n t e s t (31, 45). The t e s t depends on the f a c t that the i n t a c t plasma membrane excludes dyes such as trypan b l u e , but damaged c e l l s are s t a i n e d , p a r t i c u l a r l y i n t e n s e l y i n the nucleus. U n f o r t u n a t e l y , t h i s a c t u a l l y measures s t r u c t u r a l i n t e g r i t y , not v i a b i l i t y per se. Other t e s t s have been used i n a d d i t i o n , i n c l u d i n g e l e c t r o n microscopy, the content of adenine n u c l e o t i d e s , the a c t i v i t i e s o f v a r i o u s enzymes, and the a b i l i t y to s y n t h e s i z e v a r i o u s compounds. I t seems to t h i s reviewer that one must e s t a b l i s h f o r h i s own type o f experiments whether the c e l l s are v i a b l e using c r i t e r i a p a r t i c u l a r l y a p p l i c a b l e to the type o f r e s e a r c h being conducted. For example, i n a metabolism study one might choose a p a r t i c u l a r s u b s t r a t e which could be used as a p o s i t i v e c o n t r o l i n a l l p r e p a r a t i o n s , whose metabolism could be c o n v e n i e n t l y measured and q u a n t i f i e d f o r comparison between v a r i o u s p r e p a r a t i o n s . I t i s somewhat s u r p r i s i n g to r e p o r t that no s t u d i e s have yet been p u b l i s h e d i n v o l v i n g the metabolism o f a p e s t i c i d e i n i s o l a t e d hepatocytes. However, the technique has been widely ap p l i e d i n s t u d i e s of drug metabolism and s e v e r a l r e p o r t s on the metabolism o f a i r p o l l u t a n t s and i n d u s t r i a l chemicals have now appeared. Examples o f drug metabolism s t u d i e s are the r e p o r t s o f B i l l i n g s et al. (33) on α-1-acetylmethadol, propoxyphene, butamoxane, ethinimate, 8-methoxybutamoxane, and p - n i t r o p h e n o l ; E r i c k s o n and Holtzman (39) on ethylmorphine; Hayes and Brendel (42) on q u i n i n e s u l f a t e , dansylamide, and a n t i p y r i n e ; and Aarbakke et al. (30) on a n t i p y r i n e . In g e n e r a l , the drugs stud i e d were metabolized by the i s o l a t e d hepatocytes by N- and 0demethylation, aromatic and a l i p h a t i c h y d r o x y l a t i o n , and s u l f a t e and g l u c u r o n i c a c i d conjugation. These s t u d i e s show that the metabolism of drugs i n i s o l a t e d hepatocytes c o r r e l a t e s with in vivo drug metabolism b e t t e r than does the l i v e r homogenate 9000^ supernatant or microsomal f r a c t i o n . The r e s u l t s obtained from hepatocytes were more comparable t o l i v e r p e r f u s i o n s than t o sub c e l l u l a r f r a c t i o n s i n terms o f the r e l a t i v e r a t e s of i n d i v i d u a l r e a c t i o n s , which were sometimes f a s t e r and sometimes slower i n hepatocytes than i n microsomes. Aromatic hydrocarbons are r e a d i l y hydroxylated by i s o l a t e d hepatocytes (34, 35, 43). Bock and co-workers showed that naph thalene was converted to 1-naphthol and to 1 , 2 - d i h y d r o - l , 2 - d i hydroxynaphthalene and i t s s u l f a t e and g l u c u r o n i c a c i d conjugates (34). The i s o l a t e d hepatocytes were more e f f i c i e n t i n c a r r y i n g out these conversions than microsomes, the reason being that the enzymes r e s p o n s i b l e , mixed f u n c t i o n oxygenase, epoxide hydratase, and g l u c u r o n y l t r a n s f e r a s e , are a l l l o c a t e d i n the same mem branes. Benzo(a)pyrene was converted i n t o arene oxides, phenols,



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quinones, and d i h y d r o d i o l s as i n i t i a l products and l a t e r s u l f a t e , glucuronide, and g l u t a t h i o n e conjugates were i s o l a t e d (35). The monohydroxylated compounds and s u l f a t e e s t e r s accumulated i n t r a c e l l u l a r ^ , the 4,5- and 7,8-dihydrodiols were d i s t r i b u t e d evenl y between the c e l l s and the medium, and the 9,10-dihydrodiol accumulated i n the medium. In these experiments s i g n i f i c a n t amounts o f r a d i o a c t i v i t y were bound i r r e v e r s i b l y to c e l l u l a r macromolecules (43). The s e q u e n t i a l formation o f metabolites by i s o l a t e d hepatocytes i s i l l u s t r a t e d by s t u d i e s on the 4-hydroxylation o f b i phenyl and the subsequent conjugation o f the metabolite (44). F i g u r e 3 i l l u s t r a t e s the f a c t that h y d r o x y l a t i o n preceded g l u curonide formation and that the removal o f 4-hydroxybiphenyl by conjugation was necessary to s t i m u l a t e a second phase o f hydroxylation. Fry et at. (41) have compared i s o l a t e d c e l l s from r a t l i v e r and kidney i n terms o f t h e i r a b i l i t y to metabolize ethoxycoumarin, b i p h e n y l , benzo(a)pyrene, 4-methylumbelliferone, and benzoic a c i d . The l e v e l o f metabolism i n the kidney c e l l suspension was extremely low compared with l i v e r c e l l s f o r i n i t i a l o x i d a t i v e r e a c t i o n s , but the p a t t e r n and extent o f conjugations were very s i m i l a r between the two types o f c e l l s . The d i f f e r e n c e i n the a b i l i t y o f these c e l l s t o metabolize these compounds may r e l a t e to a d i f f e r e n t s p e c i f i c i t y o f the r e n a l cytochrome P-450, which i s not adapted to x e n o b i o t i c metabolism. The a b i l i t y t o r e t a i n a c t i v e cytochrome P-450 i n i s o l a t e d hepatocytes i s c r u c i a l t o the use o f t h i s system to study xenob i o t i c metabolism. Cytochrome P-450 d e c l i n e s r a p i d l y i n i s o l a t e d hepatocytes, but r e c e n t l y i t was discovered that the a d d i t i o n o f c e r t a i n hormones t o a primary hepatocyte c u l t u r e r e t a i n e d the cytochrome P-450 a c t i v i t y at optimum l e v e l s f o r up to 24 hours (36). These workers used primary*hepatocyte c u l t u r e s der i v e d from collagenase p r e p a r a t i o n with added hormones t o study the metabolism o f a f l a t o x i n B^. Although i s o l a t e d hepatocytes have not as yet been extens i v e l y used i n metabolism s t u d i e s , they c l e a r l y o f f e r great promise i n t h i s area. Conclusion When one compares the v a r i o u s in vitro techniques used t o study the f a t e o f x e n o b i o t i c s i n mammals, one must be impressed by the f a c t that no one o f the methods a v a i l a b l e i s adequate to a complete understanding o f the metabolism o f a compound. However, when a l l methods are taken together, a r a t h e r complete p i c t u r e can be assembled. Using c a r b a r y l , one o f the most e x t e n s i v e l y studied p e s t i c i d e s , as an example, one can see that the t o t a l in vitro r e s u l t s r a t h e r completely m i r r o r in vivo metabolism. In f a c t , c e r t a i n metabolites found in vitro have not been i s o l a t e d f o l l o w i n g in vivo s t u d i e s , the N-glucuronide noted from c e l l




144

«

Biochimica et Biophysica Acta

Figure 3. Rate of formation of 4-hydroxy biphenyl and its glucuronide conjugate in viable isolated hepatocytes. The rates of formation of (O), 4-hydroxy biphenyl and (U)> Ms glucuronide conjugate are average rates for the respective 5-min intervab. Each point represents the mean of three values obtained from different experiments (± S.D.) (44).


MENZER


4.

Intact Animal

Cells,

Tissues, and

145

Organs

c u l t u r e s . The p r i n c i p a l m e t a b o l i t e , 1-naphthol, i s found i n a l l systems s t u d i e d . The 5,6-dihydro-5,6-diol, 5-hydroxy, 1,5-diol sequence i s observed i n c e l l c u l t u r e s , while the 4-hydroxy metab o l i t e i s observed i n perfused organs and c e l l c u l t u r e s . The 1,4d i o l was a l s o i s o l a t e d from c e l l c u l t u r e s as were s e v e r a l g l y c o s i d e conjugates. One might compare the r e l a t i v e merits o f the f o u r techniques considered i n t h i s paper, adding microsomes to complete the p i c t u r e , from f o u r p o i n t s o f view: (1) ease of p r e p a r a t i o n , (2) f l e x i b i l i t y o f experimental use, (3) r e p r o d u c i b i l i t y o f r e s u l t s , and (4) the extent to which the r e s u l t s obtained m i r r o r in vivo r e s u l t s (Table I ) . Table I. R e l a t i v e merits o f in vitro techniques f o r studying the metabolism o f x e n o b i o t i c s .

r+

Ο

2ί H'

Ο

Η ο (Λ Ο

Β

CD (Λ

Η

3

(Λ (Λ

"Ο Φ Η

Favorable, p o s i t i v e aspect o f t h i s Neutral Negative aspect o f t h i s technique

C Φ

tn

t-h

t—»

C (Λ Η· Ο 3

Ease o f P r e p a r a t i o n Requirement f o r S p e c i a l Equipment Requires s p e c i a l t r a i n i n g o f personnel Time f o r p r e p a r a t i o n Expense F l e x a b i l i t y o f Experimental Use R e p r o d u c i b i l i t y o f Results M i r r o r s in vivo r e s u l t s + ο -

CD Ο*

OQ

-

Η· Ο Φ «Λ

Φ t—' h-*

η

c

Κ-* Γι C

Η Φ

+

0

SC CD »Ί3 Ρ Γ+

ο Ο Χ

Γ+

Φ (Λ

0

ο

+ +

ο +

+

+

-

+

ο +

ο

+ +

+ +

ichnique

Although the assignment o f values f o r each category i s a r b i t r a r y and s u b j e c t i v e , one must conclude from t h i s e x e r c i s e that each technique w i l l be u s e f u l f o r some purposes i n some r e s e a r c h e r ' s hands. U l t i m a t e l y our understanding o f the f a t e o f x e n o b i o t i c s i n mammals w i l l be enhanced by accumulating data from a l l sources and a p p l y i n g each b i t o f data to o b t a i n the complete p i c t u r e . Acknowledgement S c i e n t i f i c A r t i c l e No. A2519 C o n t r i b u t i o n No. 5551 o f the Maryland A g r i c u l t u r a l Experiment S t a t i o n , Department of Entomology.


146


The author thanks Miss Figen Un111 f o r her a s s i s t a n c e i n the pre p a r a t i o n o f t h i s paper and Dr. Judd 0. Nelson f o r h i s h e l p f u l advice i n i t s development.


Abstract Techniques considered include isolated whole living cells; cell, organ, and tissue culture; organ slices; and isolated per fused organs. Whole cell techniques serve as an important link between studies using purified enzymes and subcellular fractions and studies using whole organisms. The use of cells, tissues, and organs in culture is growing since they allow the researcher to explore the nature of metabolites and to research an under standing of the mechanisms of metabolism taking place within the cells or organs without the complicating regulatory influences of the whole organism. Both primary cells in culture and establish ed cell lines have been used to study xenobiotic degradation. Conversely, the effects of xenobiotics on the cell can also be conveniently studied. The combination of the two types of stud ies allows one to ascertain whether the cell's metabolism of a xenobiotic is accomplished by a healthy cell or as the result of or in combination with some cellular defect. The use of whole organs, such as perfused liver, provides the opportunity to ex tend a metabolism experiment over a longer period of time than is possible with either subcellular fractions or isolated cells. Whole organs or organ slices allow the introduction of a higher degree of cellular organization and differentiation than the single cell, but without the complications of external regulation. The use of isolated hepatocytes is a recent innovation in the study of xenobiotic metabolism and should be most useful. Literature Cited Perfused Organs 1.

Blase, B. W., and Loomis, T. A. Toxicol. Appl. Pharmacol. (1976) 37, 481. 2. Dalbey, W., and Bingham, E. Toxicol. Appl. Pharmacol. (1978) 43, 267. 3. Fuhremann, T. W., Lichtenstein, E. P., Zahlten, R. Ν., Stratman, F. W., and Schnoes, H. K. Pestic. Sci. (1974) 5, 31. 4. Law, F. C. P., Eling, T. W., Bend, J. R., and Fouts, J. R. Drug Metab. Disp. (1974) 2, 433. 5. Mehendale, H. M. Toxicol. Appl. Pharmacol. (1976) 36, 369. 6. Mehendale, H. M., and El-Bassiouni, Ε. A. Drug Metab. Disp. (1975) 3, 543. 7. Miller, L. L., Bly, C. G., Watson, M. L., and Bale, W. F. J. Exp. Med. (1951) 94, 431.



4.

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Intact Animal

Cells,

Tissues, and

Organs

147

8. Niemeier, R. W., and Bingham, E. Life Sci. (1972) 11, part II, 807. 9. Orton, T. C., Anderson, M. W., Pickett, R. D., Eling, T. C., and Fouts, J. R. J. Pharmacol. Exp. Therap. (1973) 186, 482. 10. Popov, Τ. Α., and Kagan, Y. S. Gig. Sanit. (1977) 40. [CA 87:904] 11. Ritchie, H. D., and Hardcastle, J. C. (eds.) "Isolated Organ Perfusion", University Park Press, Baltimore, 1973. 12. Ross, B. D., "Perfusion Techniques in Biochemistry", Clarendon Press, Oxford, 1972. Tissue

Slices

13. Fang, S. C., Fallin, E. and Freed, V. H. Toxicol. Appl. Pharmacol. (1973) 25, 493. 14. Fenwick, M. L., Barron, J. R. and Watson, W. A. Biochem. J. (1957) 65, 58. 15. Krebs, Η. Α., and Henseleit, K. Hoppe-Seyler's Ζ. Physiol. Chem. (1932) 210, 33. 16. O'Neil, J. J., Sanford, R. L., Wasserman, S., and Tierney, D. F. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. (1977) 43, 902. 17. Pekas, J. C. Am. J. Physiol. (1971) 220, 2008. 18. Pritchard, J. B. Toxicol. Appl. Pharmacol. (1976) 38, 621. 19. Warburg, O. Biochem. Z. (1923) 142, 317. Cell Culture 20. Baron, R. L., and Locke, R. K. Bull. Environ. Contam. Toxicol. (1970) 5, 287. 21. Locke, R. K., Bastone, V. B., and Baron, R. L. J. Agr. Food Chem. (1971) 19, 1205. 22. Huang, Ε. Α., Lu, J. Y., and Chung, R. A. Biochem. Pharmac. (1970) 19, 637. 23. Lin, Τ. Η., North, Η. Η., and Menzer, R. E. J. Agr. Food Chem. (1975) 23, 253. 24. Lin, Τ.Η.,North, Η. Η., and Menzer, R. E. J. Agr. Food Chem. (1975) 23, 257. 25. Lin, Τ. Η., Menzer, R. Ε., and North, H. H. J. Agr. Food Chem. (1976) 24, 756. 26. Muecke, W., Menzer, R. Ε., Alt, K. O., Richter, W., and Esser, H. O., Pestic. Biochem. Physiol. (1976) 6, 430. 27. North, Η. Η., and Menzer, R. E. Pestic. Biochem. Physiol. (1972) 2, 278. 28. North, Η. Η., and Menzer, R. E. J. Agr. Food Chem. (1973) 21, 509. 29. Spalding, J. W., Ford, E., Lane, D., and Blois, M. Biochem. Pharmacol. (1971) 20, 3185.


148



Isolated Hepatocytes 30. Aarbakke, J., Bessesen, Α., and Morland, J. Acta Pharmacol. Toxicol. (1977) 41, 225. 31. Baur, Η., Kasperek, S., and Pfaff, E. Hoppe-Seyler's Ζ. Physiol. Chem. (1975) 356, 827. 32. Bellemann, P., Gebhardt, R., and Mecke, D. Anal. Biochem. (1977) 81, 408. 33. Billings, R. Ε., McMahon, R. Ε., Ashmore, J., and Wagle, S. R. Drug Metab. Disposition (1977) 5, 518. 34. Bock, K. W., VanAckeren, G., Lorch, F., and Birke, F. W. Biochem. Pharmacol. (1976) 25, 2351. 35. Burke, M. D., Vodi, H., Jernström, B., and Orrenius, S. J. Biol. Chem. (1977) 252, 6424. 36. Decad, G. M., Hsieh, D. P. H., and Byard, J. L. Biochem. Biophys. Res. Commun. (1977) 78, 279. 37. Dickson, A. J., and Langslow, D. R. Biochem. Soc. Trans. (1975) 3, 1034. 38. Dickson, A. J., and Langslow, D. R. Biochem. Soc. Trans. (1977) 5, 983. 39. Erickson, R. R., and Holtzman, J. L. Biochem. Pharmacol. (1976) 25, 1501. 40. Fry, J. R., Jones, C. Α., Wiebkin, P., Bellemann, P., and Bridges, J. W. Anal. Biochem. (1976) 71, 341. 41. Fry, J. R., Wiebkin, P., Kao, J., Jones, C. Α., Gwynn, J., and Bridges, J. W. Xenobiotica (1978) 8, 113. 42. Hayes, J. S., and Brendel, K. Biochem. Pharmacol. (1976) 25, 1495. 43. Jones, C. Α., Moore, B. P., Cohen, G. Μ., Fry, J. R., Biochem. Pharmacol. (1978) 27, 693. 44. Jones, R. S., Mendis, D., and Parke, D. V. Biochim. Biophys. Acta (1977) 500, 124. 45. Seglen, P. O. "Methods in Cell Biology", D. M. Prescott (ed.), Vol. 8, Academic Press, New York, 1976, pp. 29-83. 46. Wagle, S. R. Life Sci. (1975) 17, 827. 47. Wagle, S. R., and Ingebretsen, Jr., W. R. Methods in Enzymol. (1975) 35, 579. RECEIVED

December 2 0 , 1978.


Techniques for Studying the Metabolism of Xenobiotics by Intact

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