Role of Metabolic Activation in Chemical-Induced Tissue Injury

8 Role of Metabolic Activation in Chemical-Induced

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 28, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

Tissue Injury SIDNEY D. NELSON, MICHAEL R. BOYD, and JERRY R. M I T C H E L L Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, MD 20014

An important result of metabolism studies in recent years has been the realization that many chemical compounds are metabolized by the liver and various other tissues to potent alkylating and arylating intermediates (1-12). Such studies demonstrate how chemically stable compounds can produce serious tissue lesions in man and experimental animals, including hepatic, renal, and pulmonary necrosis, bone marrow aplasia, neoplasia and other injuries. Although these lesions are rare, such toxic effects are of great c l i n i c a l concern because they often lead to irreversible failure of the liver, lungs, kidneys or other organs, and subsequent death of the patient. Many of the initial concepts of metabolic activation were developed during studies of chemical carcinogenesis; the work of the Millers in the United States (1,2) and of Magee and co-workers in England (3) has been especially illuminating. The realization that the enzyme pathways responsible for the conversion of certain chemicals to proximate carcinogens are the same microsomal mixed-function oxygenases that metabolize most drugs and other xenobiotics led to the concept that drug-induced tissue lesions might also be mediated through the covalent binding of reactive metabolites (6-11). The lack of reactivity of most chemically stable compounds and the frequent localization of tissue damage only in those organs or to those animal species having the necessary drug-metabolizing enzymes supported this view. Additionally, these studies frequently demonstrated a role for sulfhydryl-containing compounds, particularly glutathione, in protecting tissues from such toxic reactions. Most drugs and foreign compounds that enter the body are converted to chemically stable metabolites that are readily excreted into urine and b i l e , or are expired. Thus, it has become important to distinguish those toxicities that are mediated by chemically reactive metabolites and those reactions due to an exaggerated therapeutic effect or unwanted secondary effect caused by the drug or one of i t s stable metabolites. The toxicologic activity produced by the latter class of reactions usually can be monitored by 155 Jerina; Drug Metabolism Concepts ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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DRUG M E T A B O L I S M C O N C E P T S


measuring the c o n c e n t r a t i o n of the compound and i t s m e t a b o l i t e s i n body f l u i d s * However, when the response i s t i s s u e damage caused by the covalent b i n d i n g of c h e m i c a l l y r e a c t i v e m e t a b o l i t e s t o t i s s u e macromolecules, r a r e l y can a r e l a t i o n s h i p between t i s s u e l e v e l s of the m e t a b o l i t e and the s e v e r i t y of the l e s i o n be d e t e r mined* Indeed, h i g h l y r e a c t i v e m e t a b o l i t e s may e x i s t f o r o n l y a few seconds o r l e s s and w i l l t h e r e f o r e never accummulate i n body fluids. Parameters f o r s t u d y i n g r e a c t i v e m e t a b o l i t e s . How then can the formation of such c h e m i c a l l y u n s t a b l e and r e a c t i v e m e t a b o l i t e s be studied? Based on s t u d i e s where an animal model has been developed f o r a p a r t i c u l a r chemical-induced t i s s u e l e s i o n , a r e l a t i o n s h i p can o f t e n be made between the s e v e r i t y of the t i s s u e l e s i o n and the amount of m e t a b o l i t e t h a t i s c o v a l e n t l y bound to the damaged t i s s u e . That i s , covalent b i n d i n g of the r e a c t i v e m e t a b o l i t e can be used as an index of formation of the m e t a b o l i t e . Furthermore, t h i s parameter might w e l l be the most r e l i a b l e e s t i mate of the a v a i l a b i l i t y of the m e t a b o l i t e i n s i t u f o r causing t i s s u e damage, s i n c e much of the m e t a b o l i t e o f t e n decomposes or i s f u r t h e r metabolized before i t can be i s o l a t e d i n body f l u i d s . Thus, one approach t o the problem i s t o determine whether r a d i o l a b e l e d drugs administered t o animals over a wide dose range are c o v a l e n t l y bound t o macromolecules i n t a r g e t t i s s u e s t h a t subsequently become n e c r o t i c . Pretreatment of animals w i t h inducers o f drug metabolism, such as phénobarbital, or w i t h i n h i b i t o r s of drug metabolism, such as p i p e r o n y l butoxide, c o b a l t c h l o r i d e , o r ©(-naphthylisothiocyanate, s i m i l a r l y should a l t e r the r a t e of metabolism of t o x i n , the extent of covalent b i n d i n g of r e a c t i v e m e t a b o l i t e , and the s e v e r i t y of t i s s u e i n j u r y . I n c o n j u n c t i o n w i t h these s t u d i e s i n animals, experiments can be performed i n v i t r o w i t h microsomal enzymes i s o l a t e d from the t a r g e t organ t i s s u e . Covalent b i n d i n g of r e a c t i v e m e t a b o l i t e s may be one u s e f u l index of product formation when v a r i o u s a d d i t i o n s or d e l e t i o n s from the system are made, or when animals are p r e t r e a t e d w i t h v a r i o u s enzyme inducers and i n h i b i t o r s . Another u s e f u l index of r e a c t i v e product formation i n t h i s system i s the t r a p p i n g of e l e c t r o p h i l i c intermediates w i t h a l t e r n a t e n u c l e o p h i l e s such as c y s t e i n e or g l u t a t h i o n e . S t r u c t u r a l e l u c i d a t i o n of such i n t e r m e d i a t e s may o f t e n p r o v i d e i n s i g h t i n t o the s t r u c t u r e o f the i n i t i a l r e a c t i v e m e t a b o l i t e . U l t i m a t e l y , i s o l a t i o n and s t r u c t u r e e l u c i d a t i o n of the r a d i o l a b e l e d m a t e r i a l bound to the t i s s u e macromolecules (RNA, DNA, p r o t e i n ) can be c a r r i e d out. The approach d e s c r i b e d has been used to i m p l i c a t e t o x i c m e t a b o l i t e s as mediators of the t o x i c i t i e s caused by s e v e r a l drugs. Hepatic n e c r o s i s has been a s s o c i a t e d w i t h the use of hydrazides i s o n i a z i d ( I ) , a t u b e r c u l o s t a t i c agent, and i p r o n i a z i d ( I I ) , an a n t i d e p r e s s a n t . Both h e p a t i c and r e n a l i n j u r y are a s s o c i a t e d w i t h the use of h i g h doses of two s u b s t i t u t e d aminophenol a n a l g e s i c s , acetaminophen ( I I I ) and phenacetln ( I V ) . The f u r a n - c o n t a i n i n g d i u r e t i c agent, furosemide (V), and the thiophene-containing

Jerina; Drug Metabolism Concepts ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

157

a n t i b i o t i c , c e p h a l o r i d i n e ( V I ) , are a s s o c i a t e d w i t h r e n a l i n j u r y i n man. Ipomeanol ( V I I ) , a f u r a n - c o n t a i n i n g d e r i v a t i v e produced by moldy sweet potatoes, i s an example o f a chemical t o x i n which produces pulmonary l e s i o n s v i a r e a c t i v e m e t a b o l i t e formation. These and other experimental s t u d i e s w i t h model compounds w i l l be presented t o i l l u s t r a t e the concepts which u n d e r l i e the r o l e o f metabolic a c t i v a t i o n i n chemical-induced t i s s u e i n j u r y and the parameters used t o e s t a b l i s h these concepts.


Hydrazines and Hydrazides I s o n i a z i d . A good example of t o x i c drug r e a c t i o n s caused by metabolic a c t i v a t i o n i s i s o n i a z i d - i n d u c e d l i v e r i n j u r y . This drug provides a unique o p p o r t u n i t y to show how a study can be pursued from a c l i n i c a l l y manifest t i s s u e l e s i o n t o the proposal o f a r a t i o n a l chemical mechanism f o r the t o x i c i t y . C l i n i c a l f i n d i n g s . Three c l i n i c a l s t u d i e s (13-15) provided evidence t h a t metabolic a c t i v a t i o n was i n v o l v e d i n the s e r i o u s h e p a t i t i s caused by i s o n i a z i d when t h i s drug was administered i n t h e r a p e u t i c doses. F i r s t was a p r o s p e c t i v e study c a r r i e d out i n 1972 (13). SGOT and serum b i l i r u b i n concentrations were examined monthly i n 250 p a t i e n t s r e c e i v i n g i s o n i a z i d f o r one year. These b i o c h e m i c a l i n d i c e s i n d i c a t e d that i s o n i a z i d was hepatotoxic i n a l a r g e p r o p o r t i o n o f i n d i v i d u a l s but most adapted t o the i n s u l t and recovered r a t h e r than developing severe h e p a t i t i s . Measurement of plasma concentrations o f i s o n i a z i d i n these p a t i e n t s , f a i l e d t o show a c o r r e l a t i o n between plasma l e v e l s o f i s o n i a z i d and l i v e r i n j u r y . I n t h i s study, no a n t i - i s o n i a z i d a n t i b o d i e s were found and no c o r r e l a t i o n was seen between h e p a t i c i n j u r y and a n t i n u c l e a r a n t i b o d i e s measured a t the end o f the study. The seoncd study was a r e t r o s p e c t i v e a n a l y s i s o f 114 p a t i e n t s w i t h i s o n i a z i d - r e l a t e d h e p a t i t i s (14). Some o f the important f i n d i n g s were t h a t : 1) i s o n i a z i d - r e l a t e d l i v e r i n j u r y was c l i n i c a l l y i n d i s t i n g u i s h a b l e b i o c h e m i c a l l y and m o r p h o l o g i c a l l y from i p r o n i a z i d - i n d u c e d l i v e r damage or from other causes o f acute h e p a t o c e l l u l a r i n j u r y such as v i r a l h e p a t i t i s ; 2) no c l i n i c a l evidence such as r a s h , f e v e r , a r t h r a l g i a s o r e o s i n o p h i l i a was found f o r h y p e r s e n s i t i v i t y mechanism; 3) about 30% o f the p a t i e n t s w i t h h e p a t i c r e a c t i o n s were r e s i d e n t s o f Honolulu and o f O r i e n t a l a n c e s t r y ; on g e n e t i c b a s i s , 90% or more o f these p a t i e n t s would be expected t o be r a p i d a c e t y l a t o r s o f i s o n i a z i d i n c o n t r a s t t o b l a c k and w h i t e populations i n whom 45% are r a p i d a c e t y l a t o r s (16). I n the t h i r d study (15), 21 n o n - O r i e n t a l p a t i e n t s who had recovered from i s o n i a z i d h e p a t i t i s were g e n e t i c a l l y phenotyped as r a p i d o r slow a c e t y l a t o r s o f i s o n i a z i d u s i n g the sulfamethazine method. E i g h t y - s i x percent o f them d i s p l a y e d the r a p i d a c e t y l a t o r phenotype f o r i s o n i a z i d metabolism. Metabolism s t u d i e s i n man. Based on these c l i n i c a l f i n d i n g s , we re-examined the metabolism of i s o n i a z i d and i d e n t i f i e d the



158


m e t a b o l i t e s by co-chromatography, reverse i s o t o p e d i l u t i o n w i t h s y n t h e s i z e d standards and by mass s p e c t r a l a n a l y s i s (15). T r i t i u m r i n g - l a b e l e d i s o n i a z i d and a c e t y l i s o n i a z i d , the major primary m e t a b o l i t e of i s o n i a z i d , were administered to human v o l u n t e e r s i n s i n g l e 300 mg doses and u r i n a r y m e t a b o l i t e s were c o l l e c t e d f o r 24 h r s . As shown i n F i g u r e 2, about 55% of a dose of a c e t y l i s o n i a z i d was metabolized by h y d r o l y s i s t o i s o n i c o t i n i c a c i d and f r e e a c e t y l h y d r a z i n e r e g a r d l e s s of g e n e t i c phenotype of the p a t i e n t s f o r a c e t y l a t i n g i s o n i a z i d * I n c o n t r a s t , p a t t e r n of m e t a b o l i t e s a f t e r a d m i n i s t r a t i o n of i s o n i a z i d was very dependent upon the r a t e a t which i s o n i a z i d was a c e t y l a t e d * On the b a s i s of the r e l a t i v e amounts of a c e t y l i s o n i a z i d and i s o n i c o t i n i c a c i d excreted i n t o the u r i n e , we c a l c u l a t e d t h a t almost a l l of the i s o n i c o t i n i c a c i d was formed by way of a c e t y l i s o n i a z i d * We a l s o c a l c u l a t e d that p a t i e n t s who were f a s t m e t a b o l i z e r s of i s o n i a z i d converted about 94% of an i s o n i a z i d dose to a c e t y l i s o n i a z i d ; o n l y 2.8% of the drug was exc r e t e d unchanged i n the u r i n e and 3.6% as hydrazone conjugates. Slow a c e t y l a t o r s , on the other hand, excreted almost 37% of the drug i n the u r i n e e i t h e r f r e e or as a hydrazone. Thus, o n l y 63% was converted t o a c e t y l i s o n i a z i d and subsequently to i s o n i c o t i n i c a c i d and a c e t y l h y d r a z i n e . We concluded, t h e r e f o r e , that f a s t a c e t y l a t o r s are exposed t o much more a c e t y l i s o n i a z i d and a c e t y l hydrazine than are slow a c e t y l a t o r s . Hepatic n e c r o s i s i n animals. A c e t y l i s o n i a z i d and i s o n i a z i d were given t o r a t s , mice and hamsters to see i f they could produce h e p a t i c n e c r o s i s (17,18). These hydrazines were given i n a doseresponse manner to s e v e r a l hundred animals. I s o n i a z i d d i d not cause n e c r o s i s i n any of the animals. However, a c e t y l i s o n i a z i d produced o c c a s i o n a l s i n g l e c e l l n e c r o s i s i n r a t s and mice. Moreover, as shown i n Table I , pretreatment of r a t s w i t h phénobarbital, which i s known to i n c r e a s e drug m e t a b o l i z i n g enzymes, g r e a t l y p o t e n t i a t e d the n e c r o s i s . The l i v e r damage was prevented by p r e treatment of r a t s w i t h c o b a l t c h l o r i d e , which i n h i b i t s s y n t h e s i s of cytochrome P-450 m e t a b o l i z i n g enzymes. S i m i l a r l y when hydrol y s i s of a c e t y l i s o n i a z i d was i n h i b i t e d by pretreatment of r a t s w i t h b i s - p a r a - n i t r o p h e n y l phosphate (BNPP), the n e c r o s i s was prevented. The e f f e c t on the l i v e r of the h y d r o l y s i s product, a c e t y l h y d r a z i n e , was t h e r e f o r e examined. This hydrazine i s a very potent hepatotoxin which produces h e p a t i c n e c r o s i s i n phenobarbital-pret r e a t e d r a t s a f t e r s i n g l e doses of 10 mg/kg. The n e c r o s i s was p o t e n t i a t e d by pretreatment w i t h phénobarbital and prevented by pretreatment w i t h c o b a l t c h l o r i d e (Table I ) . However, BNPP, which i n h i b i t e d the h y d r o l y s i s of a c e t y l i s o n i a z i d and prevented the n e c r o s i s , had no e f f e c t on n e c r o s i s produced by a c e t y l h y d r a z i n e . Thus, the metabolic a c t i v a t i o n of the l i b e r a t e d a c e t y l h y d r a z i n e moiety of a c e t y l i s o n i a z i d to a t o x i c m e t a b o l i t e s a t i s f a c t o r i l y accounts f o r the h e p a t i c n e c r o s i s produced by i s o n i a z i d . Subsequently, i s o n i a z i d i t s e l f was shown to produce acute h e p a t i c n e c r o s i s i n p h e n o b a r b i t a l - t r e a t e d r a t s . The p r o p o r t i o n or the i s o n i a z i d that i s a c e t y l a t e d i n r a t s decreases markedly


8.

NELSON

C-N-NH

Chemical-Induced

ETAL.

S H H

Tissue

Injury

159

/C 3 H

C-N-N-CH

2

Ô

ô

un

(I)

9

HN-C-CH

HN—C—CH

5


3

0-CH -CH

OH (III)

2

3

(IV)

HOOC-"

HOOC

OAc

(VI)

(V)

CH,^CH,

HO'' ΓΗ

Figure 1. Structures of the compounds discussed in the text

(VII)

X Of DOSE DRUG

PATIENTS ACETYLATIOtl RATE 00

AcIMH

FAST (2)

AcINH

INH HTDRAZONES

AcINH

INA DERIVATIVES

ESTIMATED ACETYL HYDRAZINE

...

54.912.2

45.112.7

45.112.7

...

SLOW (3)

—

53.811.2

46.211.1

46.211.1

...

INH

FAST (3)

2.810.4

3.610.4

49.211.9

44.413.9

41.013.8

3.410.1

INH

SLOW (4)

10.910.8 26.5±4.8

32.111.2

30.513.5

26.813.3

3.7*0.2

INH

ESTIMATED HYDRAZINE

JLS-CCH CH C-

o -

3

ACETYLISONIAZID

ISONIAZID

ISONIAZID HÏDRAZONES

ACETYLHYDRAZINB

ISONICOTINIC ACID

Figure 2. Twenty-four hour urinary excretion of metabolites after administration of 300 mg of acetylisoniazid- H-ring-labeled (AcINH) or isoniazid- H-ring-labeled (INH) to male volunteers (See Ref. 15) s

3



160


Table I ACUTE HEPATIC NECROSIS IN RATS PRODUCED BY ISONIAZID (INH), ACETYLISONIAZID (AcINH), ACETYLHYDRAZINE (AcHz), IPRONIAZID (IpINH), AND ISOPROPYLHYDRAZINE (IpHz)

Treatments

INH 100 mg/kg*

AcINH 200 mg/kg

AcHZ 20 mg/kg

0 or +

IpINH 200 mg/kg

IpHz 20 mg/kg

+

+

None

0

0 or +

Phénobarbital

+

++

-H+

Phénobarbital + CoCl

0

0

0

0 or+

0 or +

Phénobarbital + BNPP

0

0

-H-+

0 or+

+4+4-

2

+++

*Admlnisterd every hour for 6 hours* +CoCl • cobalt chloride. 2

ÎBNPP • bls-para-nitrophenyl phosphate.


8.

NELSON

ET AL.

Chemical-Induced

Tissue

Injury

161


above 100 mg/kg i n d i c a t i n g a s a t u r a b l e mechanism. Thus, a s i n g l e l a r g e dose does not cause l i v e r n e c r o s i s , but the a d m i n i s t r a t i o n o f i s o n i a z i d i n s i x s i n g l e doses o f 100 mg/kg per hour caused acute h e p a t i c n e c r o s i s (Table I ) . Covalent b i n d i n g s t u d i e s i n v i v o . As f u r t h e r support f o r the hypothesis t h a t a c e t y l i s o n i a z i d i s converted i n the b o d y j j o a c h e m i c a l l y r e a c t i v e form v i a a c t i v a t i o n o f a c e t y l h y d r a i n e , Ca c e t y l i s o n i a z i d r a d i o l a b e l e d i n the a c e t y l moiety and C - a c e t y l hydrazine were given t o r a t s and evidence f o r covalent b i t i d i n g t o t i s s u e macromolecules was sought (18,19). A l a r g e amount o f cov a l e n t b i n d i n g was found upon d i g e s t i o n o f the p r o t e i n s i n the l i v e r , the t a r g e t organ f o r t o x i c i t y , but l i t t l e was found i n other t i s s u e s . This b i n d i n g was p r o p o r t i o n a l t o dose, was i n creased by pretreatment w i t h phénobarbital and was markedly decreased by pretreatment w i t h c o b a l t c h l o r i d e (Table I I ) . However, no c o v a l e n t l y bound r a d i o l a b e l e d m a t e r i a l was found when a c e t y l i s o n i a z i d r a d i o l a b e l e d i n the p y r i d i n e r i n g was administered. Thus, the r e a c t i v e m e t a b o l i t e came only from the a c e t y l h y d r a z i n e moiety. BNPP, which b l o c k s the h y d r o l y s i s o f a c e t y l i s o n i a z i d , decreased the covalent b i n d i n g o f ^ C - a c e t y l i s o n i a z i d |> t h a t o f C - a c e t y l h y d r a z i n e , p a r a l l e l i n g the e f f e c t o f BNPP on the hepatic necrosis. ut n

o

t

14

Covalent b i n d i n g s t u d i e s i n v i t r o . Based on the e f f e c t s o f mixed f u n c t i o n oxygenase inducers and i n h i b i t o r s on the h e p a t i c n e c r o s i s and covalent b i n d i n g found i n animals, experiments were c a r r i e d out u s i n g l i v e r microsomes i n v i t r o t o determine t h e enzyme requirements f o r the b i n d i n g r e a c t i o n . The r e s u l t s o f experiments w i t h a c e t y l h y d r a z i n e and r a t l i v e r microsomes under v a r i o u s c o n d i t i o n s (Table I I I ) showed t h a t a s u b s t a n t i a l amount o f covalent b i n d i n g occurred a t 37 C i n the presence o f l i v e r microsomes, a i r and NADPH. The b i n d i n g was almost a b o l i s h e d by l a c k o f NADPH, heat dénaturât i o n o f the enzymes, o r l a c k o f oxygen. A carbon monoxide: oxygen atmosphere, SKF-525A, p i p e r o n y l butoxide pretreatment, o r an antibody a g a i n s t NADPH cytochrome £ reductase i n h i b i t e d covalent b i n d i n g , thereby i n d i c a t i n g i n v o l v e ment o f a cytochrome P-450 mixed f u n c t i o n oxygenase. Furthermore, experiments w i t h h e p a t i c microsomes prepared immediately f o l l o w i n g the traumatic death o f a h e a l t h y young a d u l t male demonstrate t h a t the a c t i v a t i o n system i s present i n human t i s s u e s (19, Table III). — G l u t a t h i o n e and c y s t e i n e , n a t u r a l l y o c c u r r i n g s u l f h y d r y l compounds, s u b s t a n t i a l l y decreased covalent b i n d i n g i n v i t r o by formation o f the adducts, S - a c e t y l g l u t a t h i o n e and N - a c e t y l c y s t e i n e . The work o f Smith and G o r i n (21), which showed t h a t S - a c e t y l c y s t e i n e rearranges r a p i d l y a t n e u t r a l pH values t o the thermodynamically more s t a b l e N - a c e t y l c y s t e i n e , suggests t h a t the i n i t i a l product might have been S - a c e t y l c y s t e i n e which subsequently r e arranged t o the observed product, N - a c e t l y c y s t e i n e . Both N - a c e t y l c y s t e i n e and S - a c e t y l g l u t a t h i o n e were i s o l a t e d from e


162

DRUG M E T A B O L I S M

CONCEPTS

Table I I EFFECT OF TREATMENTS ON IN VIVO HEPATIC COVALENT BINDING OF 3

3

ISONIAZID- H-RING-LABELED (INH),* ACETYLISONIAZID- H-RING LABELED 14

(AcINH)*, ACETYLISONIAZID- C-ACETYL-LABELED (AçINH), ACETYL-HYDRA14

3

ZINE- C-ACETYL-LABELED (AcHz), IPRONIAZID- H-RING-LABELED

(IpINH),


3

IPR0NIAZID-2- H-IS0PR0PYL-LABELED (ΙηΙΝΗ), and ISOPROPYLHYDRAZINE3

2- H-IS0PR0PYL-LABELED (IpHz) IN RATS. R e s u l t s a r e expressed as means + standard e r r o r s o f 3 separate experiments u s i n g 3 animals i n each experiment.

Treatment

AcINH IpINH IDINH AcHz IpHz 200 200 20 200 20 mg/kg mg/kg mg/kg mg/kg mg/kg Covalent B i n d i n g Covalent B i n d i n g nmole/mg p r o t e i n nmole/mg p r o t e i n (6 h r a f t e r dose) (6 h r a f t e r dose)

None

0.20 + .021

0.15 + .012

0.09 + .015

0.28 + .029

0.35 + .023

Pb**

0.31 + .021

0.19 + .012

0.10 + .015

0.53 + .038

0.44 + .038

0.15 + .039

0.09 + .008

0.18 + .017

0.22 + .029

0.11 + .033

0.23 + .035

0.17 + .019

0.32 + .025

Pb + C o C l

2

Pb + ΒΝΡΡΦ

Covalent b i n d i n g f o r these two compounds was p r o t e i n f o r a l l treatments. + CoCl

β 2

< 0.05 nmole/mg

phénobarbital + c o b a l t c h l o r i d e

tpb + BNPP » phénobarbital + b i s - p a r a - n i t r o p h e n y l phosphate


8.

NELSON

ET

AL.

Chemical-Induced

Tissue

Injury

163

Table I I I 14

14

COVALENT BINDINGQIN VITRO OF ACETYL-( C)-HYDRAZINE ( C-AcHz) AND ISOPROPYL-(2-nQ-HYDRAZINE CH-IpHz) TO RAT LIVER MICROSOMES A

Conditions

X-AcHz (1 mM)

~Ή-ΙρΗζ (0.1 mM)

** Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 28, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

% of C o n t r o l A.

C o n t r o l * ( a i r atmosphere) B o i l e d microsomes - C o f a c t o r (NADPH generating system) +NADH (-NADPH generating system) 100% N atmosphere 2

6%

10%

7% 15%

7% 11%

atmosphere

92%

97%

C0:0

(9:1) atmosphere

37%

48%

64% 25% 35% 49%

70% 45% 58% 65%

2

: 0

2

+SKF 525-A (0.2 mM) P i p e r o n y l butoxide+ +GSH (1 mM) +Cysteine (1 mM)

C.

100% 12%

2

N

B.

100% 13%

$

Preimmune - g l o b u l i n

0.099

nmoles/mg/15 min 0.328

Immune Ï - g l o b u l i n (NADPH-cytochrome c reductase antibody)

0.048

0.159

C o n t r o l ( a i r atmosphere) - c o f a c t o r (NADPH generating system)

0.16

Human Microsomes 0.37

0.02

0.03

Microsomes were prepared from r a t l i v e r and human l i v e r , incubated as d e s c r i b e d i n Table IV and covalent b i n d i n g was determined ( 1 9 ) . The c o n t r o l b i n d i n g o f AcHz w i t h r a t l i v e r microsomes was 0.55 nmoles/mg/15 min and f o r IpHz was 0.58 nmoles/mg/15 min. ^Administered

(0.3 ml) i . p . 30 min p r i o r t o s a c r i f i c i n g

the animal.

"^Each i n c u b a t i o n contained 7 mg of p a r t i a l l y p u r i f i e d preimmune o r immune jf - g l o b u l i n per mg microsomal p r o t e i n , as p r e v i o u s l y described (20).


164



i n c u b a t i o n mixtures by g e l f i l t r a t i o n on Sephadex f o l l o w e d by anion exchange chromatography* The products were then c h a r a c t e r i z e d by chemical i o n i z a t i o n mass spectrometry (22,23)* I p r o n i a z i d * Studies i n animals o f the metabolism of i p r o n i a z i d ( I I ) , an antidepressant drug removed from c l i n i c a l use because o f a h i g h i n c i d e n c e of h e p a t i t i s s i m i l a r to t h a t o f i s o n i a z i d r e v e a l e d t h a t i p r o n i a z i d a l s o r e q u i r e d enzymatic h y d r o l y s i s to produce the h e p a t i c l e s i o n (Table I ) . S p e c i f i c r a d i o l a b e l l n g and covalent b i n d i n g s t u d i e s showed that i s o p r o p y l h y d r a z i n e was r e leased by h y d r o l y s i s and then o x i d a t i v e l y a c t i v a t e d i n v i t r o to a potent hepatotoxin (19, Table I I ) . M e t a b o l i c a c t i v a t i o n of i s o p r o p y l h y d r a z i n e , the hepatotoxic m e t a b o l i t e of i p r o n a z i d , t o a r e a c t i v e intermediate showed enzyme requirements v i r t u a l l y i d e n t i c a l t o those f o r the a c t i v a t i o n of a c e t y l h y d r a z i n e (Table I I I ) . Thus, a cytochrome P-450 oxygenase mediated the covalent b i n d i n g o f i s o p r o p y l h y d r a z i n e to t i s s u e p r o t e i n . Trapping experiments w i t h c y s t e i n e and g l u t a t h i o n e showed t h a t S - i s o p r o p y l c y s t e i n e and S - i s o p r o p y l g l u t a t h i o n e were formed (23)· The a c t i v a t i n g enzyme system could be assessed k i n e t i c a l l y u s i n g covalent b i n d i n g o f r a d i o l a b e l e d m e t a b o l i t e as an index o f r e a c t i v e product formation* A d o u b l e - r e c i p r o c a l p l o t of the enzyme-dependent b i n d i n g o f a c e t y l h y d r a z i n e to microsomal p r o t e i n s ( F i g u r e 3A) shows t h a t the r e a c t i o n r a t e i s markedly increased by phénobarbital pretreatment, which p o t e n t i a t e d the h e p a t i c n e c r o s i s and b i n d i n g i n v i v o , whereas i t i s decreased by pretreatment o f the animals w i t h c o b a l t c h l o r i d e , which blocked the h e p a t i c n e c r o s i s and b i n d i n g i n v i v o . The same e f f e c t s were found f o r the b i n d i n g r e a c t i o n o f i s o p r o p y l h y d r a z i n e to r a t l i v e r microsomes, except that the Κ f o r b i n d i n g was 1/10 that f o r a c e t y l h y d r a z i n e (Figure 3B). This may account f o r the greater h e p a t o t o x i c i t y observed w i t h i s o p r o p y l hydrazine when compared to t h a t of a c e t y l h y d r a z i n e . I n a d d i t o n , the e v o l u t i o n o f propane from microsomal r e a c t i o n s was determined by gas chromatography and gas chromatography-mass spectrometry .As shown i n Table IV, phénobarbital i n c r e a s e d both covalent b i n d i n g and propane formation, whereas c o b a l t c h l o r i d e decreased both. Double-isotope experiments w i t h a c e t y l i s o n i a z i d - a c e t y l h y d r a z i n e and i p r o n i a z i d - i s o p r o p y l h y d r a z i n e . In order to study the metabolic a c t i v a t i o n process f o r the covalent b i n d i n g of a c e t y l i s o n i a z i d and a c e t y l h y d r a z i n e i n more d e t a i l , we administered to r a t s a mixture o f a c e t y l i s o n i a z i d and a mixture of a c e t y l h y d r a z i n e l a b e l e d w i t h t r i t i u m and carbon-14 i n the a c e t y l moiety. The H/ r a t i o o f the c o v a l e n t l y bound m e t a b o l i t e from e i t h e r a c e t y l i s o n i a z i d or a c e t y l h y d r a z i n e was almost i d e n t i c a l to the ^H/^C r a t i o o f the administered mixture (19, Table V ) . This i n d i c a t e d 3r ij y ! group was bound. Moreover, Incubation of the ^H- and ^ C - a c e t y l h y d r a z i n e w i t h r a t l i v e r microsomes i n v i t r o gave the same r e s u l t s (19, Table V ) . 3

t h a t

t h e

e n t

e

a c e t


8.

Chemical-Induced

NELSON E T A L .

Tissue

Injury

165

200

PHENOBARBITAL + COBALTOUS CHLORIDE PRETREATMENT K^-0.98 aM V^-0.03 NMOLES/MG/MIN

150 1


(NMOLES/MG/MIN)"

NORMAL 1^-0.95^ V ax-0.06 NMOLES/MG/MIN B

PHENOBARBITAL 1^-1.13 «M V - _ - 0 . l l NMOLES/MG/MIN

1

1/S («Μ" ) 100

r

PHENOBARBITAL + CoCl, Κ - 0.07 m V ? " 0.04 naolM/ag/aln

NORMAL K^- 0.09 WÊL V_ • 0.07 naol*s/ag/aln

PHENOBARBITAL K_« 0·10 «M - 0.13 naoUs/ae/aln

-15

-10

-5

Figure S. Lineweaver-Burk plots of mixed function oxidase-dependent covalent binding of acetylhydrazine (A) ana isopropylhydrazine (B) to rat microsomal protein in vitro. For each incubation, rat microsomes were prepared, incubated under air with either Cacetylhydrazine (A) or isopropyl-2 ( H)-hydrazine (B) and a NADPH-generating system, and covalent binding was determined. 14

s


166


CONCEPTS


TABLE IV CORRELATION OF PROPANE EVOLUTION WITH IN VITRO COVALENT BINDING OF ISOPROPYL-(2- H)-HYDRAZINE TO HEPATIC MICROSOMES Ice-cold incubation mixtures (3 ml) contained rat l i v e r microsomal protein (2 mg/ml) isolated from rats pretreaÇed as i n dicated; phosphate buffer, pH 7.4, 83 mM; isopropyl-(2- H)hydrazine, 0.1 mM; and a NADPH-generating system (NADP, 0.64 mM; glucose-6-phosphate, 15.5 mM; glucose-6-phosphate dehydrogenase, 2U/ml; MgC^, 10 mM). Reactions were incubated under a i r i n septum-sealed incubation vessels for 15 min with shaking (Dubnoff shaker incubator) at 37 C and covalent binding determined (19). The head-space gases were analyzed by GLC as described i n Ref. 19} the propane effluent was trapped in Aquasol s c i n t i l l a n t cooled i n dry ice-acetone, and radioa c t i v i t y was counted by s c i n t i l l a t i o n spectrometry. Results are expressed as means + standard deviations. Numbers i n parentheses are number of determinations.

Treatment

None

I n v i t r o Covalent B i n d i n g (nmoles/mg p r o t e i n / 1 5 min)

0.58 + 0.051 (9)

Phénobarbital (75 mg/kg i.p.

χ 4 days)1.06 + 0.059 (6)

Phénobarbital (75 mg/kg i.p. χ 4 days) + cobalt chloride (30 mg/kg s.c. 12 hourly χ 4 doses) 0.33 + 0.006 (6)

Propane evolved (% o f t o t a l radioactivity i n 15 min)

13.0% + 1.00 (9)

19.5% + 0.75 (6)*

9.4% + 0.81 (6)*

*P jC 0.05 when compared to respective control values as determined by Student's t test.


8.

Chemical-Induced

NELSON E T A L .

Tissue

Injury

167

Table V 3

14

RATIOS ( H / C ) RADIOLABEL BOUND TO HEPATIC PROTEIN VERSUS THAT IN INITIAL SUBSTRATE MIXTURES Mixtures o f

14 3 C-carbonyl- and H-methyl-labeled t/

i s o n i a z i d (AcINH; 200 mg/kg; sp. a c t .

acetyl*x

C, 0.15 mCi/mmole;

H,

0.53 mCi/mmole) and s i m i l a r mixtures o f a c e t y l h y d r a z i n e (AcHz, Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 28, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch008

1 4

3

20 mg/kg; sp. a c t . C , 0.36 mCi/mmole; H , 1.01 mCi/mmole) were administered t o male F i s c h e r r a t s . I n a d d i t i o n , mixtures o f 14 3 s p e c i f i c a l l y l a b e l e d i s o p r o p y l - ( 2 - C ) - and i s o p r o p y l - ( 2 - H ) l a b e l e d i p r o n i a z i d (IpINH. 200 mg/kg; sp. a c t C , 0.50 mCi/ 1 4

3

mmole; sp. a c t .

H, 1.43 mCi/mmole) and i s o p r o p y l h y d r a z i n e 14 3 (IpHz, 20 mg/kg; sp. a c t . C, 0.30 mCi/mmole; sp. a c t . H,

0.87 mCi/mmole) were administered t o F i s c h e r r a t s . I n other experiments, mixtures o f H- and C-acetylhydrazine (1 mM) 3 14 and i s o p r o p y l - ( 2 - H)- and i s o p r o p y l - ( 2 - C ) - hydrazine (0.1 mM) were incubated i n a i r w i t h an NADPH-generating system and w i t h microsomes i s o l a t e d from F i s c h e r r a t l i v e r .

Covalent b i n d i n g o f

r a d i o l a b e l t o h e p a t i c t i s s u e p r o t e i n was determined by methods p r e v i o u s l y d e s c r i b e d (19) and found t o be 0.20 nmoles/mg ( i n v i v o , AcHz), 0.28 nmoles/mg ( i n v i t r o , IpHz). 3

Values a r e r e p o r t -

4

ed as H/^* C r a t i o s o f the c o v a l e n t l y bound r a d i o l a b e l , as determined by the c h a n n e l s - r a t i o method u s i n g i n t e g r a l counting, 3

14

d i v i d e d by the H / C r a t i o o f the i n i t i a l s u b s t r a t e mixture as determined by the same method. R e s u l t s a r e expressed as means + standard e r r o r s o f 4 such determinations.

Conditions

Substrate

AcINH

AcHz

IpINH

IpHz

In vivo (6 h r a f t e r dosing) 0.90 + .055 0.92+0.021 0.92+. 032 0.96+0.060 In v i t r o (15 min i n c u b a t i o n s )

-

0.94+0,012

-

0.98+0.022



168


CONCEPTS

L i v e r microsomes and NADPH were a l s o incubated w i t h c y s t e i n e and approximately equimolar amounts o f a c e t y l - and t r i d e u t e r o a c e t y l h y d r a z i n e (22). N - a c e t y l c y s t e i n e was i s o l a t e d from i n cubation mixtures c o n t a i n i n g NADPH, oxygen, c y s t e i n e and a c e t y l hydrazine. Chemical i o n i z a t i o n mass spectrometry showed q u a s i molecular i o n s (QM ) a t m/e 164 and 167 f o r the non- and t r i d e u t e r a t e d N - a c e t y l c y s t e i n e . These i o n s were monitored and found t o have t h e same H/D r a t i o as t h e quasimolecular ions o f the a c e t y l hydrazine s u b s t r a t e mixture (m/e 75 and 78, F i g u r e 4 ) . This study e l i m i n a t e d ketene as the r e a c t i v e i n t e r m e d i a t e . S i m i l a r experiments w i t h i p r o n i a z i d and i s o p r o p y l h y d r a z i n e , l a b e l e d w i t h t r i t i u m and carbon-14 i n the methine carbon o f the i s o p r o p y l group, showed t h a t e q u i v a l e n t amounts o f t r i t i u m and carbon-14 were bound both i n v i v o and i n v i t r o , demonstrating t h a t methine hydrogen was r e t a i n e d and t h e r e f o r e e l i m i n a t i n g acetone as t h e intermediate i n the b i n d i n g r e a c t i o n (19). T h i s was confirmed by a t w i n - i o n study u s i n g s p e c i f i c a l l y C-2 deuterated i s o p r o p y l h y d r a z i n e . The r e a c t i v e m e t a b o l i t e was trapped from microsomal i n c u b a t i o n s i n v i t r o w i t h c y s t e i n e . Mass s p e c t r a l a n a l y s i s o f t h e i s o l a t e d c y s t e i n e d e r i v a t i v e showed t h a t S - i s o p r o p y l c y s t e i n e was formed and t h a t no deuterium was l o s t from the i s o p r o p y l group (23). M e c h a n i s t i c i m p l i c a t i o n s f o r the metabolic a c t i v a t i o n o f t o x i c m e t a b o l i t e s o f i s o n i a z i d and i p r o n i a z i d . From the r e s u l t s i n v i v o showing c o r r e l a t i o n s between t i s s u e n e c r o s i s and covalent b i n d i n g , s t u d i e s i n v i t r o showing a microsomal P-450 oxygenase requirement, and t r a p p i n g experiments w i t h c y s t e i n e and g l u t a t h i o n e , we propose the f o l l o w i n g r e a c t i o n scheme (Figure 5) f o r formation o f t o x i c m e t a b o l i t e s from i s o n i a z i d and i p r o n i a z i d . I s o n i a z i d i s a c e t y l a t e d t o i t s major m e t a b o l i t e a c e t y l i s o n i a z i d . I n man, r a p i d a c e t y l a t o r s convert a t l e a s t 35% more i s o n i a z i d t o a c e t y l i s o n i a z i d than slow a c e t y l a t o r s . A c e t y l i s o n i a z i d i s then e f f i c i e n t l y hydrolyzed t o i s o n i c o t i n i c a c i d and a c e t y l hydrazine. A c e t y l h y d r a z i n e i s f u r t h e r metabolized by a P-450 oxygenase, p o s s i b l y t o a N-hydroxy hydrazine. T h i s intermediate would probably dehydrate t o a c e t y l d i a z e n e which could be t h e e l e c t r o p h i l i c a c y l a t i n g s p e c i e s . However, mono-substituted d i a zenes are known t o fragment i n the presence o f oxygen most l i k e l y t o r a d i c a l s (24) and t h i s could be t h e t o x i c i n t e r m e d i a t e . Ketene has been e l i m i n a t e d as the r e a c t i v e a c y l a t i n g s p e c i e s o f a c e t y l hydrazine by chemical i o n i z a t i o n mass s p e c t r a l t w i n - i o n study w i t h t r i d e u t e r o - a c e t y l h y d r a z i n e , which i n d i c a t e d t h a t t h e e n t i r e a c e t y l group was bound (22). By mechanisms t h a t are not understood t h e r e a c t i v e m e t a b o l i t e i n i t i a t e s processes t h a t l e a d t o h e p a t i c necrosis. I p r o n i a z i d i s hydrolyzed t o i s o n i c o t i n i c a c i d and i s o p r o p y l hydrazine. Isopropylhydrazine i s then f u r t h e r o x i d i z e d t o a r e a c t i v e a l k y l a t i n g agent. Since the e v o l u t i o n o f propane p a r a l l e l s covalent b i n d i n g , we suspect t h e two r e a c t i o n s d e r i v e from common


NELSON E T A L .

Chemical-Induced

Injury

0AC75

100

I

Tissue

S

ΐθΜ,+78

80

Η R-C-N-NH

2

R-CH *CD3 3


60

•Ι

40 20

100

90

80 M/E

70

100 Ο H Il H I R-C-N-C-CH2-SH

δ

1 C

80

QM+167

COOH R=CH +CD 3

60

3

40 '22 UO 121 1 2 3 |

20

1

80

100

120 M/E

3

3

140

160

180

Figure 4. Chemical ionization mass spectra (isobutane reactant gas) of a sample of the substrate mixture of acetyl- and trideuteroacetylhydrazine (A) and of the cysteine adduct iso lated from a microsomal incubation containing the substrate mixture, NADPH and cysteine (B) (See Ref. 22)



170


CONCEPTS

i n t e r m e d i a t e . Furthermore, a t w i n - i o n study w i t h s p e c i f i c a l l y C-2 deuterated i s o p r o p y l h y d r a z i n e , s i m i l a r t o the study c a r r i e d out with trideuteroacetylhydrazine, indicated that the e n t i r e isopro p y l group was r e t a i n e d i n t h e bound m e t a b o l i t e . A r e a c t i o n scheme ( F i g u r e 5 ) compatible w i t h these r e s u l t s i s the formation o f the i s o p r o p y l r a d i c a l o r c a t i o n from i s o p r o p y l d i a z e n e . These r e a c t i v e a l k y l a t i n g agents then c o v a l e n t l y b i n d t o t i s s u e macromolecules. Whatever t h e intermediate may be, i t i s c l e a r t h a t o x i d a t i v e a c t i v a t i o n o f these hydrazines by microsomal enzymes mediates l i v e r n e c r o s i s i n animals. Since these enzymes a r e present i n human l i v e r t i s s u e , these intermediates probably cause the s e r i o u s and o c c a s i o n a l l y l e t h a l h e p a t i t i s seen w i t h i s o n i a z i d and i p r o n i a z i d therapy i n man.

t Covalent Binding to Macromolecules Λ

Propane

* •

Expired Propane

Hepatic Necrosis

Figure 5.

Proposed metabolic activation pathways for isoniazid, acetyliso niazid, and isopropylisoniazid (iproniazid)


8.

NELSON

ET AL.

Chemical-Induced

Tissue Injury

171


Acetaminophen S t u d i e s i n v i v o i n man and l a b o r a t o r y animale» Acetaminophen ( p - h y d r o x y a c e t a n i l i d e , I I I ) i s a commonly used m i l d a n a l g e s i c which i s a p p a r e n t l y q u i t e s a f e when taken i n normal t h e r a p e u t i c doses. However, l a r g e overdoses cause l i f e - t h r e a t e n i n g l i v e r l e s i o n s i n man (2J5,26), r a t s (27,28), mice (28), and hamsters (29). P r i o r treatment o f animals w i t h inducers o f drug metabolism, such as phénobarbital o r 3-methylcholanthrene, g r e a t l y p o t e n t i a t e s t h e s e v e r i t y o f t h e n e c r o s i s (28,30). I n c o n t r a s t , pretreatments w i t h i n h i b i t o r s o f drug metabolism, such as p i p e r o n y l b u t o x i d e , c o b a l t c h l o r i d e , o r «-naphthylisothiocyanate, prevent t h e n e c r o s i s (28,30). A l a c k o f c o r r e l a t i o n between acetaminophen t i s s u e l e v e l s and acetaminophen-induced h e p a t i c n e c r o s i s i n d i c a t e s t h a t a t o x i c m e t a b o l i t e r a t h e r than acetaminophen i t s e l f causes the h e p a t i c t i s s u e i n j u r y . Acetaminophen r a d i o l a b e l e d w i t h t r i t i u m o r w i t h carbon-14 was g i v e n t o normal mice and mice p r e t r e a t e d w i t h compounds t h a t a l t e r e d acetaminophen-induced h e p a t i c n e c r o s i s . The animals were k i l l e d a t v a r i o u s times and t h e l i v e r s examined f o r c o v a l e n t l y bound m e t a b o l i t e s o f acetaminophen. Autoradiograms showed covalent b i n d i n g o f acetaminophen p r e f e r e n t i a l l y i n t h e n e c r o t i c c e n t r i l o b u l a r area o f t h e l i v e r , i . e . , there was a d i r e c t c o r r e l a t i o n between the two measurable parameters t i s s u e n e c r o s i s and c o v a l e n t b i n d i n g (31). Pretreatment w i t h an inducer o f microsomal metabolism, phénobarbital, i n c r e a s e d b i n d i n g , whereas p r e t r e a t ments w i t h d i f f e r e n t i n h i b i t o r s o f metabolism decreased b i n d i n g . Thus, t h e e f f e c t o f treatment on covalent b i n d i n g c o r r e l a t e d d i r e c t l y w i t h treatment e f f e c t s on h e p a t i c n e c r o s i s . Evidence f o r the c o v a l e n t nature o f t h e b i n d i n g was obtained by d i g e s t i o n o f s o l v e n t - e x t r a c t e d l i v e r p r o t e i n w i t h protease and i s o l a t i o n o f the r a d i o l a b e l bound t o amino a c i d and peptide fragments. These s t u d i e s i n d i c a t e d t h a t acetaminophen was converted by microsomal enzymes, t o a r e a c t i v e a r y l a t i n g agent which c o v a l e n t l y bound t o macromolecules i n the t a r g e t t i s s u e f o r damage, t h e l i v e r . Concept o f a dose-threshold f o r t o x i c i t y . Because o f t h e s t r i k i n g c o r r e l a t i o n between t h e s e v e r i t y o f h e p a t o t o x i c i t y and the extent o f covalent b i n d i n g by t h e a r y l a t i n g m e t a b o l i t e o f acetaminophen, i t was s u r p r i s i n g t h a t s i g n i f i c a n t b i n d i n g d i d not occur u n t i l over 60% o f t h e drug had been e l i m i n a t e d from t h e l i v e r . G l u t a t h i o n e i s depleted from t h e l i v e r o f animals r e c e i v i n g acetaminophen because i t combines w i t h a minor m e t a b o l i t e o f the drug and forms a r e a d i l y excreted mercapturic a c i d (4,30,32, 33). Thus t h e p o s s i b i l i t y a r i s e s t h a t t h e a r y l a t i n g m e t a b o l i t e o f acetaminophen i n t i a l l y i s d e t o x i f i e d by r e a c t i n g p r e f e r e n t i a l l y w i t h g l u t a t h i o n e (Figure 6 ) . A f t e r t h e major r o u t e s o f acetaminophen metabolism ( s u l f a t i o n and g l u c u r o n i d a t i o n pathways) become s a t u r a t e d , and a f t e r t h e l i v e r i s depleted o f g l u t a t h i o n e , t h e r e a c t i v e m e t a b o l i t e can combine w i t h l i v e r macromolecules and by undefined mechanisms cause c e l l death.



172

CONCEPTS

ACETAMINOPHEN


HNCOCH

HNCOCH,

HHCOCH-

3

0

OH P-450 MIXEDJFUNCTION OXIDASE HO-NCOCH " 3

•

NCOCH,

0 -

POSTULATED TOXIC INTERMEDIATES

NUCLEOPHILIC CELL MACROMOLECULES HNCOCH,

HNCOCH,

OLUTATHIONE

0 =

CELL MACROMOLECULES

•

ERCAPTURIC ACID

• CELL DEATH

Figure 6. Pathways of acetaminophen metabolism



8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

173

I n support o f t h i s view, covalent b i n d i n g and l i v e r n e c r o s i s occurred o n l y a f t e r doses o f acetaminophen s u f f i c i e n t l y l a r g e t o exceed t h e a v a i l a b i l i t y o f g l u t a t h i o n e f o r d e t o x i f i c a t i o n (Figure 7). S i m i l a r l y , when g l u t a t h i o n e concentrations i n the l i v e r were compared w i t h t h e extent o f covalent b i n d i n g a t v a r i o u s times a f t e r t h e a d m i n i s t r a t i o n o f acetaminophen, s i g n i f i c a n t b i n d i n g had occurred o n l y a f t e r g l u t a t h i o n e was s e v e r e l y depleted (30,32). I n accord w i t h t h i s view, p r i o r a d m i n i s t r a t i o n o f d i e t h y l maleate, which decreases t h e g l u t a t h i o n e c o n c e n t r a t i o n i n l i v e r without causing l i v e r n e c r o s i s , markedly p o t e n t i a t e s t h e l i v e r damage caused by acetaminophen (30,32), and d i e t s t h a t lower the conc e n t r a t i o n o f g l u t a t h i o n e enhance the t o x i c i t y as w e l l (34). On the other hand, the a d m i n i s t r a t i o n o f the a l t e r n a t e s u l f h y d r y l compounds, c y s t e i n e o r cysteamine, prevented t h e l i v e r n e c r o s i s (6,32). Recent s t u d i e s w i t h acetaminophen have supported t h e view t h a t a g l u t a t h i o n e t h r e s h o l d i s o p e r a t i v e i n man as w e l l as l a b o r a t o r y animals (33.35,36). Therefore, s u l f h y d r y l reagents such as c y s t e i n e , cysteamine, d i m e r c a p r o l , and g l u t a t h i o n e i t s e l f a r e being s u c c e s s f u l l y used i n the therapy o f acetaminophen-overdosed p a t i e n t s (37). T h i s emphasizes t h e importance o f understanding b i o c h e m i c a l mechanisms o f t o x i c i t y before r a t i o n a l approaches t o treatment can be made. Phenacetin Phenacetin ( p - e t h o x y a c e t a n i l i d e , IV) has been i m p l i c a t e d i n r e n a l i n j u r y i n man (38). Therefore, we considered t h e p o s s i b i l i t y t h a t t h i s s p e c i a l type o f n e p h r i t i s , c a l l e d a n a l g e s i c nephropathy, was r e l a t e d t o metabolic a c t i v a t i o n . Although no c o n s i s t e n t l y r e p r o d u c i b l e l e s i o n could be obtained i n l a b o r a t o r y animals t r e a t e d w i t h l a r g e doses o f phenacetin, l i v e r n e c r o s i s was observed, e s p e c i a l l y i n hamsters (39)» a species u n u s u a l l y s u s c e p t i b l e t o acetaminophen-induced h e p a t i c n e c r o s i s (29,30). As w i t h acetaminophen, phenacetin-induced l i v e r n e c r o s i s i n hamsters i s p o t e n t i a t e d by pretreatment w i t h 3-methylcholanthrene but not by phénobarbital. For example, phenacetin doses o f 400 mg/kg produce massive c e n t r i l o b u l a r n e c r o s i s i n 3-methylcholanthrene-treated animals. Moreover, t h e s e v e r i t y o f n e c r o s i s p a r a l l e l s t h e magnitude o f t h e covalent b i n d i n g o f r a d i o l a b e l e d phenacetin t o h e p a t i c p r o t e i n s and t h e d e p l e t i o n o f h e p a t i c g l u t a t h i o n e (39). L i t t l e b i n d i n g o r h e p a t i c n e c r o s i s occurs a t doses that deplete h e p a t i c g l u t a t h i o n e l e s s than 80%. However, c o n s i d e r a b l e b i n d i n g and n e c r o s i s occur at doses t h a t deplete g l u t a t h i o n e more than 80%. Pretreatment o f hamsters w i t h 3-methylcholanthrene i n c r e a s e s d e p l e t i o n o f h e p a t i c g l u t a t h i o n e , t h e covalent b i n d i n g , and the s e v e r i t y o f n e c r o s i s a f t e r phenacetin, whereas pretreatment w i t h cobaltous c h l o r i d e o r p i p e r o n y l butoxide decreases them. These f i n d i n g s i n d i c a t e t h a t g l u t a t h i o n e i n t h e l i v e r prevents covalent b i n d i n g and n e c r o s i s by combining w i t h a r e a c t i v e a r y l a t i n g m e t a b o l i t e o f phenacetin.




IN v m

2* SO OOSC OF^-ACCTAMINOPMCNfmc/kt,

·?

CONCEPTS

*»·

I.p.)

S 5

*

as

so

OOSt OF ^N-ACtTAMIMOPNtN f • I S / M l !·»·>

Figure 7. Relationship in vivo between hepatic glutathione concentration, the formation of an acetaminophen-glutathione conjugate (measured in unne as acetaminophen mercapturic acid), and covalent binding of an acetaminophen metabolite to liver proteins (See Ref. 33)



8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

175

M e c h a n i s i t i c I m p l i c a t i o n s f o r the metabolic a c t i v a t i o n o f acetaminophen and phenacetin based on s t u d i e s u s i n g hamster microsomal enzymes* Although the i d e n t i t y of the a r y l a t i n g m e t a b o l i t e s o f acetaminophen and phenacetin are u n c e r t a i n , the involvement o f N-hydroxy d e r i v a t i v e s o r arene oxides as r e a c t i v e m e t a b o l i t e s has been p o s t u l a t e d (30, 40). Experiments i n v i t r o w i t h hamster l i v e r microsomes suggests t h a t the a r y l a t i n g m e t a b o l i t e s o f a c e t aminophen and phenacetin are d i f f e r e n t , a t l e a s t i n t h i s microsoma l system (41)* The evidence i s as f o l l o w s : l ) T h e maximum v e l o c i t y of covalent b i n d i n g f o r phenacetin exceeds t h a t f o r acetaminophen, showing t h a t phenacetin i s not f i r s t deethylated to acetaminophen which i s then a c t i v a t e d * 2)Pretreatment o f hamsters w i t h 3-methylcholanthrene i n c r e a s e s the r a t e o f covalent b i n d i n g f o r acetaminophen but decreases the r a t e of b i n d i n g f o r phenacetin* 3) Phénob a r b i t a l pretreatment i n c r e a s e s the r a t e o f covalent b i n d i n g f o r phenacetin without a f f e c t i n g the r a t e of b i n d i n g f o r acetaminophen* 4) When covalent b i n d i n g was prevented by t r a p p i n g o f the r e a c t i v e m e t a b o l i t e s w i t h g l u t a t h i o n e d u r i n g i n c u b a t i o n s c a r r i e d out under atmospheres o f oxygen-18, r e d u c t i o n by Raney-nickel o f the g l u t a t h i o n e conjugates formed from e i t h e r acetaminophen or phenacetin y i e l d e d acetaminophen; however, the acetaminophen conjugate formed d u r i n g i n c u b a t i o n w i t h phenacetin i n c o r p o r a t e d 50% oxygen-18 i n t o the 4 - p o s i t i o n , whereas the acetaminophen-glutathione conjugate d u r i n g i n c u b a t i o n w i t h acetaminophen i n c o r p o r a t e d v i r t u a l l y no oxygen-18. Mechanisms based on these r e s u l t s are presented i n F i g u r e 8. The l a c k of i n c o r p o r a t i o n of oxygen-18 i n t o the g l u t a t h i o n e conjugate d e r i v e d from acetaminophen i s c o n s i s t e n t w i t h e i t h e r an N-hydroxylation o r 2,3-epoxidation mechanism. I n d i r e c t evidence i n v i v o supports an N-hydroxy l a t i o n mechanism f o r the metabolic a c t i v a t i o n of acetaminophen. Masking of the amide n i t r o g e n , as i n N-methyl-4-hydroxy a c e t a n i l i d e , b l o c k s h e p a t o t o x i c i t y (39). Pretreatment o f hamsters w i t h 3-methylcholanthrene, which i n creases the h e p a t o t o x i c i t y of acetaminophen, correspondingly i n creases the N - h y d r o x y l a t i o n of 4 - c h l o r o a c e t a n i l i d e and 2 - a c e t y l a minofluorene and the covalent b i n d i n g o f acetaminophen (20, 42, 43) By c o n t r a s t , phénobarbital pretreatment n e i t h e r a l t e r s the r a t e o f N-hydroxylation o f 4 - c h l o r o a c e t a n i l i d e nor the covalent b i n d i n g of acetaminophen (43). I t seems l i k e l y t h a t i f an N-hydroxylated m e t a b o l i t e were formed i t would subsequently undergo dehydration t o a c h e m i c a l l y r e a c t i v e imidoquinone before a r y l a t i n g t i s s u e macromolecules (Figure 8 ) . The i n c o r p o r a t i o n of 50% oxygen-18 i n t o the 4 - p o s i t i o n of the a r y l a t i n g m e t a b o l i t e of phenacetin i s p e r p l e x i n g . The s t r o n g imp l i c a t i o n i s t h a t the carbon atom i n t h i s p o s i t i o n (C-4 o f the a r o matic r i n g ) becomes t e t r a h e d r a l , b i n d i n g e q u i v a l e n t oxygen-18, der i v e d from molecular oxygen, and oxygen-16. Mechanisms c o n s i s t e n t w i t h t h i s i n t e r p r e t a t i o n are presented i n F i g u r e 8. Although these mechanisms are c o n s i s t e n t w i t h the r e s u l t s obt a i n e d i n v i t r o w i t h phenacetin, they may have l i t t l e b e a r i n g on the s i t u a t i o n i n v i v o . The major m e t a b o l i t e of phenacetin i n v i v p



176


OH

CONCEPTS

OH PHENACETIN

R R R.R-H R-R-

Figure 8.

^M^HJ'

Possible reaction mechanisms for reactive metabolite formation from acetaminophen and phenacetin using hamster liver microsomes


8.

NELSON E T A L .

Chemical-Induced

Tissue

Injury

177


i s acetaminophen and those pretreatments i n hamsters which i n crease h e p a t i c n e c r o s i s and c o v a l e n t b i n d i n g f o r acetaminophen i n c r e a s e the h e p a t i c n e c r o s i s and c o v a l e n t b i n d i n g f o r phenacetin (39). A d d i t i o n a l s t u d i e s i n v i v o w i t h phenacetin, s p e c i f i c a l l y deuterated i n theot-methylene carbon atom o f the 4-ethoxy group, a l s o i n d i c a t e d t h a t d e e t h y l a t i o n o f phenacetin t o acetaminophen i s a t least p a r t i a l l y rate-determining f o r hepatic tissue i n j u r y 45). Renal i n j u r y w i t h a c e t a n i l i d e s . R e a c t i v e i n t e r m e d i a t e s o f acetaminophen, phenacetin, and other a c e t a n i l i d e s may a l s o mediate the r e n a l i n j u r y caused by these compounds. Nery (46) has suggested t h a t the N-hydroxylated d e r i v a t i v e o f phenacetin i s an i n t e r m e d i a t e f o r minor u r i n a r y m e t a b o l i t e s o f phenacetin. Calder e t a l . (47) subsequently examined the n e p h r o t o x i c i t y o f N-hydroxyphenacetin, 4-aminophenol, hydroquinone, and p-benzoquinone and found acute r e n a l t u b u l a r n e c r o s i s i n r a t s . I t t h e r e f o r e seemed p o s s i b l e t h a t acetaminophen and phenacetin c o u l d be n e p h r o t o x i c through t h e i r N-hydroxy m e t a b o l i t e s and u l t i m a t e l y through the common r e a c t i v e d e r i v a t i v e , N-acetyl-4-benzoimidoquinone. Both acetaminophen and phenacetin c o v a l e n t l y b i n d t o a s m a l l extent t o F i s c h e r r a t kidney and acetaminophen causes r e n a l tubul a r n e c r o s i s (39). Both compounds d e p l e t e r e n a l g l u t a t h i o n e , w i t h acetaminophen much more potent than phenacetin. Experiments i n v i t r o w i t h r a t kidney microsomes show t h a t these compounds can be a c t i v a t e d t o r e a c t i v e m e t a b o l i t e s . A l t e r n a t i v e l y , acetaminophen and phenacetin may be N-hydroxylated i n the l i v e r and t r a n s ported t o the k i d n e y , p o s s i b l y as N-O-glucuronides. The g l u c u r o n i d e s might then be h y d r o l y z e d under the a c i d i c c o n d i t i o n s i n the u r i n e o r by glucuronidases i n the kidney o r u r i n e t o cause the observed r e n a l i n j u r y . However, 3-methylcholanthrene pretreatment i n c r e a s e s the h e p a t i c n e c r o s i s , but s l i g h t l y decreases the r e n a l n e c r o s i s produced by acetaminophen, suggesting t h a t the acetaminophen a c t i v a t i o n r e s p o n s i b l e f o r r e n a l i n j u r y occurs i n the kidney i t s e l f . Furans and Thiophenes Furosemide T o x i c i t y i n v i v o o f furosemide. Furosemide (V), a f r e q u e n t l y used d i u r e t i c drug, i s c o n t r a i n d i c a t e d i n pregnancy because o f i t s recognized t e r a t o g e n i c p o t e n t i a l (48). The drug a l s o has been reported t o p o t e n t i a t e r e n a l i n j u r y when used i n combination w i t h c e p h a l o r i d i n e (49, 50). Furosemide produces massive h e p a t i c n e c r o s i s i n mice and the n e c r o s i s i s prevented when metabolism i s i n h i b i t e d by pretreatment o f mice w i t h p i p e r o n y l b u t o x i d e , c o b a l t c h l o r i d e and «c-naphthylisothiocyanate (51). Covalent b i n d i n g o f the drug t o h e p a t i c t i s s u e i n mice i s a l s o blocked by these p r e treatments and occurs a few hours b e f o r e h i s t o l o g i c a l l y r e c o g n i s a b l e n e c r o s i s . Thus, the formation o f a r e a c t i v e furosemide metab o l i t e i s c a u s a l l y r e l a t e d t o the development o f furosemideinduced h e p a t i c n e c r o s i s .




178

As w i t h acetaminophen and phenacetin, the h e p a t i c n e c r o s i s produced by furosemide a l s o e x h i b i t s a dose-threshold f o r t o x i c i t y . No covalent b i n d i n g o r n e c r o s i s occurs u n t i l a dose of 100 mg/kg i s exceeded. U n l i k e the dose t h r e s h o l d f o r acetaminophen and phen e a c e t i n h e p a t o t o x i c i t y , the furosemide t h r e s h o l d i s not due t o a p r o t e c t i v e r o l e of g l u t a t h i o n e , s i n c e furosemide does not deplete h e p a t i c g l u t a t h i o n e . Studies of metabolism, d i s t r i b u t i o n , and r e v e r s i b l e plasma p r o t e i n b i n d i n g of furosemide a f t e r t o x i c and nont o x i c doses i n d i c a t e t h a t the dose-threshold f o r t o x i c i t y r e s u l t s from e i t h e r s a t u r a t i o n o f the organic anion b i n d i n g s i t e s on plasma p r o t e i n s a f t e r t o x i c doses, o r p o s s i b l y s a t u r a t i o n of b i l i a r y or r e n a l e x c r e t i o n of the drug (52). P o s s i b l e mechanism of metabolic a c t i v a t i o n . The h e p a t i c i n j u r y produced by furosemide apparently r e s u l t s from the metabolic a c t i v a t i o n of the f u r a n r i n g , p o s s i b l y by an e p o x i d a t i o n s i m i l a r t o t h a t proposed i n F i g u r e 9. Furosemide, r a d i o l a b e l e d w i t h t r i tium i n i t s f u r a n moiety, i s bound c o v a l e n t l y t o h e p a t i c microsomes i n the presence of oxygen and NADPH t o the same extent as furosemide r a d i o l a b e l e d s p e c i f i c a l l y w i t h s u l f u r - 3 5 i n i t s sulfonamide moiety, demonstrating t h a t the bound m e t a b o l i t e cont a i n s both p a r t s of the furosemide molecule. To determine where the b i n d i n g occurred on the molecule, the m e t a b o l i t e - p r o t e i n conj u g a t e s i s o l a t e d from the l i v e r were hydrolyzed under m i l d a c i d c o n d i t i o n s t h a t s p l i t furosemide i n t o i t s methylfuran and s u l f a m y o l a n t h r a n i l i c a c i d p o r t i o n s . The b i n d i n g of f u r a n - r a d i o l a b e l e d ^ r o s e m i d e t o p r o t e i n was unchanged, whereas the b i n d i n g of S-labeled furosemide was l o s t . The r e s u l t s suggested t h a t the f u r a n r i n g was being m e t a b o l i c a l l y a c t i v a t e d (51, 52). A d d i t i o n a l s t u d i e s i n v i t r o reported by W i r t h e t a l . (53) u s i n g ^ r f - H] furosemide, [35S] furosemide], k ~ H] furosemide, and H ] furosemide i n d i c a t e d t h a t formation of a p o s s i b l e e l e c t r o p h i l i c imine i n t e r m e d i a t e was u n l i k e l y and t h a t theoC-carbon was not a s i t e of metabolic a c t i v a t i o n . This f u r t h e r i m p l i c a t e d the f u r a n r i n g . Since covalent b i n d i n g was enhanced by an epoxide hydrase i n h i b i t o r and d i d not occur when tetrahydro [35S] furosemide was used as s u b s t r a t e , the authors speculated t h a t an arene oxide i n t e r m e d i a t e of the f u r a n moiety was i n v o l v e d i n the b i n d i n g . 4-Ipomeanol T o x i c i t y i n v i v o of 4-ipomeanol. Chemicals which r e p r o d u c i b l y produce an acute, s p e c i f i c pulmonary t o x i c i t y by r o u t e s of admini s t r a t i o n other than i n h a l a t i o n are r a r e and t h e i r mechanisms of a c t i o n are p o o r l y understood. 4-Ipomeanol ( V I I ) , a 3 - s u b s t i t u t e d f u r a n and the major component of "lung edema f a c t o r " produced i n sweet potatoes (Ipomoea b a t a t a s ) i n f e c t e d w i t h a common mold, has provided a v a l u a b l e t o o l w i t h which t o probe chemical-induced lung disease(54).The i n g e s t i o n of mold-damaged sweet potatoes has been i m p l i c a t e d f o r many years i n outbreaks of p o i s o n i n g i n c a t t l e . A f f e c t e d animals s u f f e r severe and o f t e n f a t a l r e s p i r a t o r y d i s t r e s s .



8.

NELSON

ET AL.

Chemical-Induced

Tissue

Injury

179

The same k i n d o f lung damage observed i n c a t t l e can be produced i n l a b o r a t o r y animals by a d m i n i s t r a t i o n o f s y n t h e t i c 4-ipomeanol (55» 56). The lung i s t h e primary t a r g e t organ i n most species* P a t h o l o g i c a l changes, such as p l e u r a l e f f u s i o n s , i n t r a a l v e o l a r and p e r i v a s c u l a r edema a r e apparent w i t h i n 6-24 hours a f t e r administration of the t o x i n . Studies were undertaken t o determine t h e p o s s i b l e formation of a c h e m i c a l l y r e a c t i v e m e t a b o l i t e i n t h i s pulmonary t o x i c i t y . Rats have been used as t h e experimental species i n a l l experiments d e s c r i b e d here. Pretreatments o f animals w i t h metabolic i n h i b i t o r s such as p y r a z o l e , p i p e r o n y l butoxide, and c o b a l t c h l o r i d e a l l markedly reduced the t o x i c i t y o f 4-ipomeanol i n the lung (57, 58). Phénobarbital, an inducer o f mixed f u n c t i o n oxygenase a c t i v i t y d i d not a l t e r t h e nature o f the lung t o x i c i t y but s i g n i f i c a n t l y increased the LD50 v a l u e , p o s s i b l y by i n c r e a s i n g d e t o x i f i c a t i o n pathways more than t o x i c pathways (59). Another type o f inducer, 3-methylcholanthrene showed a s t r i k i n g phenomenon when i t was used t o p r e t r e a t r a t s . M o r t a l i t y was decreased because o f marked decrease i n lung damage. I n c o n t r a s t , t i s s u e i n j u r y increased d r a m a t i c a l l y i n t h e l i v e r w i t h appearance o f widespread c e n t r i l o b u l a r n e c r o s i s (60). Thus, the t a r g e t organ f o r t o x i c i t y had switched from t h e lung t o the l i v e r . Covalent b i n d i n g i n v i v o and i n v i t r o . I n non-pretreated r a t s , r a d i o a c t i v i t y from 14C-4-ipomeanol becomes c o v a l e n t l y bound, p r e f e r e n t i a l l y t o t h e lungs, a f t e r a l l routes o f a d m i n i s t r a t i o n . Pretreatments w i t h mixed-function oxygenase i n h i b i t o r s , which decreased lung t o x i c i t y , caused a p a r a l l e l decrease i n covalent b i n d i n g t o lung t i s s u e i n v i v o and t o lung microsomes i n v i t r o (57, 58, 61). Phénobarbital pretreatment a l s o decreased the covalent b i n d i n g o f t o x i n t o both lung and l i v e r t i s s u e i n v i v o . However, t h e maximal r a t e o f b i n d i n g t o l i v e r microsomes i n v i t r o was increased whereas no change occurred i n b i n d i n g t o lung microsomes. The a l t e r a t i o n o f t a r g e t organ s p e c i f i c i t y f o r t i s s u e damage observed w i t h 3-methylcholanthrene pretreatment was p a r a l l e l e d by s i m i l a r a l t e r a t i o n o f covalent b i n d i n g o f r a d i o l a b e l e d 4-ipomeanol i n v i v o (60). The l e v e l o f c o v a l e n t l y bound t o x i n was markedly e l e v a t e d i n l i v e r s o f 3-methylcholanthrene-induced r a t s , whereas b i n d i n g t o lung was s i g n i f i c a n t l y reduced. The r a t e o f b i n d i n g t o l i v e r microsomes i n v i t r o was a l s o markedly i n c r e a s e d , whereas the r a t e o f b i n d i n g t o lung microsomes was unchanged (60). An important c o n c l u s i o n can be drawn from the experiments i n 3-methylcholanthrene p r e t r e a t e d animals. The t o x i c m e t a b o l i t e o f 4- ipomeanol i s so r e a c t i v e t h a t l i t t l e , i f any, o f i t escapes t h e organ i n which i t i s formed. I t t h e r e f o r e appears that i n normal animals the s p e c i f i c lung t o x i c i t y produced by 4-ipomeanol r e s u l t s p r i m a r i l y from pulmonary metabolism o f t h e agent; t h e l i v e r i s not a s i g n i f i c a n t source o f t h e r e a c t i v e m e t a b o l i t e t h a t binds t o and damages t h e lungs. The i n c r e a s e i n h e p a t i c t o x i c i t y i n 3-methylcholanthrene p r e t r e a t e d animals i s due t o an increased h e p a t i c metabolism o f 4-ipomeanol; t h e r e d u c t i o n i n pulmonary t o x i c i t y



180

CONCEPTS


probably i s due t o an e l e v a t e d r a t e o f h e p a t i c clearance o f t o x i n . Nature o f the c h e m i c a l l y r e a c t i v e m e t a b o l i t e o f 4-ipomeanol. Studies i n v i v o and i n v i t r o i n d i c a t e t h a t the r e a c t i v e m e t a b o l i t e formed by mixed f u n c t i o n oxygenase-catalyzed metabolism o f 4-ipomeanol i s a h i g h l y e l e c t r o p h i l i c species (57, 58). A d d i t i o n o f the n u c l e o p h i l i c t r i p e p t i d e , g l u t a t h i o n e , markedly i n h i b i t e d covalent b i n d i n g o f 4-ipomeanol i n v i t r o , presumably by a c t i n g as an a l t e r n a t e n u c l e o p h i l e . D e p l e t i o n of endogenous g l u t a t h i o n e i n v i v o by d i e t h y l m a l e a t e pretreatment s i g n i f i c a n t l y increased t o x i c i t y and covalent b i n d i n g o f 4-ipomeanol i n v i v o . Analogs o f 4-ipomeanol i n which the f u r a n moiety was replaced by phenyl o r methyl s u b s t i t u e n t s were not metabolized t o t o x i c e l e c t r o p h i l e s i n v i v o o r i n v i t r o (58). Thus, the furan r i n g appears t o be e s s e n t i a l f o r the observed t o x i c i t y and covalent b i n d i n g . Based on these r e s u l t s and those found f o r the metabolic a c t i v a t i o n o f furosemide, F i g u r e 10 r e v e a l s a p o s s i b l e scheme f o r f u r a n a c t i v a t i o n . Since f u r a n has l e s s a r o m a t i c i t y than benzene, i t i s not u n l i k e l y t h a t t h i s heteroaromatic nucleus could form an epoxide. T h i s epoxide would probably be q u i t e r e a c t i v e , and could y i e l d other e l e c t r o p h i l e s by spontaneous r e arrangement o r r i n g s c i s s i o n r e a c t i o n s . This arene oxide could a l s o be d e a c t i v a t e d by an epoxide hydrase, g l u t a t h i o n e , o r a g l u t a t h i o n e t r a n s f e r a s e . R e l a t i v e a c t i v i t i e s o f the v a r i o u s pathways i n t h e v a r i o u s t i s s u e s would modulate s u s c e p t i b i l i t y t o t h e toxin. H e p a t o t o x i c i t y , r e n a l t o x i c i t y , and pulmonary t o x i c i t y a r e a l s o caused by other f u r a n compounds, some of which show a g l u t a t h i o n e t h r e s h o l d , and others which show no such t h r e s h o l d (51). Furan, 2-hydroxymethylfuran, and 2 - a c e t y l f u r a n a r e hepatot o x i c . Furosemide, f u r a n , 2,3-benzofuran and c e r t a i n other furans produce acute r e n a l t u b u l a r n e c r o s i s . Other simple furans such as 2-methylfuran, 3-methylfuran, and furan i t s e l f produce lung damage, and pulmonary edema (58). Thus, a v a r i e t y of t i s s u e l e s i o n s seen a f t e r the use o f f u r a n - c o n t a i n i n g compounds probably r e s u l t s from metabolic a c t i v a t i o n s i m i l a r t o t h a t proposed f o r 4-ipomeanol and furosemide. Cephaloridine Extension o f the f u r a n s t u d i e s t o thiophene, another h e t e r o aromatic d e r i v a t i v e , has shown t h a t s e v e r a l thiophene-containing compounds produce h e p a t i c and r e n a l n e c r o s i s (39). Cephaloridine ( V I ) , a w i d e l y p r e s c r i b e d cephalosporin a n t i b i o t i c , has i t s use l i m i t e d p r i m a r i l y because o f r e n a l n e c r o s i s a s s o c i a t e d w i t h such therapy (49). Pretreatments o f mice w i t h p i p e r o n y l butoxide and c o b a l t c h l o r i d e decrease r e n a l n e c r o s i s caused by c e p h a l o r i d i n e . F u r t h e r s t u d i e s a r e i n progress t o determine i f t h i s s p e c i f i c r e n a l n e c r o s i s i s mediated by a r e a c t i v e m e t a b o l i t e .


8.

NELSON E T A L .

Chemical-Induced

F«rf«r«M«liyd«.

Tissue

Injury

181

Frmm Wmre—mié* A

Antkranilic Acid A

«lMCMr«nM«

6lucvr«ni

Role of Metabolic Activation in Chemical-Induced Tissue Injury

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