Immunohistochemical Detection of Antigenic ... - ACS Publications

Chapter 30

Immunohistochemical Detection of Antigenic Biomarkers in Microwave-Fixed Target Tissues Acetaminophen—Protein Adducts 1

1

2

Thomas J . Bucci , Alan R. Warbritton , Jack A. Hinson , and Dean W. Roberts 2

1

Pathology Associates, Inc., Jefferson, AR 72079-9502 Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079-9502

Downloaded by TUFTS UNIV on June 2, 2018 | https://pubs.acs.org Publication Date: December 26, 1990 | doi: 10.1021/bk-1990-0451.ch030

2

Immunohistochemical localization of adducts can be accomplished using antiserum developed and characterized for quantitative immunoassays. The 3-(cystein-S-yl)acetaminophen protein adduct is associated with the toxicity of the prototype hepatotoxin, acetaminophen. Immunohistochemical localization of this adduct is described to illustrate the technique as an adjunct to other methods in the assessment of toxicity. This method provides direct correlation between presence of adduct and morphologic evidence of cell injury. Microwave irradiation was pioneered as a fixation method to simultaneously preserve tissue structure and adduct antigenicity. Many chemicals that are active as toxicants or carcinogens have electrophilic properties or are metabolically converted to electrophiles which react with nucleophilic centers in nucleic acids and proteins to form covalent adducts. Covalent binding of a reactive metabolite to essential cell constituents is frequently a critical event that leads to toxicity. The complex formed by reactive metabolite binding to cell macromolecules (adduct) constitutes a novel molecular species in the organism, and it may contain unique antigenic determinants or "neoantigens". Immunological assays that recognize these neoantigens can be used to study the role of the macromolecular adduct in toxicity. Once an important neoantigen is known, antisera raised against an immunogen that contains the critical epitope may be the basis of a variety of immunoassays. Since the parent compound is usually a hapten which, by itself, is too small to be an immunogen, appropriate immunogenic conjugates must be isolated or synthesized. Antisera raised against macromolecular conjugates of the parent compound or a structurally related metabolite may recognize 1) the parent compound 2) the modified macromolecule, or 3) a unique adduct. The criteria used to This chapter not subject to U.S. copyright Published 1991 American Chemical Society Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


328

IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS

select a n a n t i s e r u m o r m o n o c l o n a l a n t i b o d y clone are d i c t a t e d b y t h e objectives of t h e i n t e n d e d a p p l i c a t i o n . T h u s , t h e u s e of h i g h l y c h a r a c t e r ized antisera is essential to meaningful interpretation. I n m a n y c a s e s , a n t i b o d i e s developed t o d e t e c t a n d q u a n t i f y t h e s e a n t i g e n i c a d d u c t s (and o t h e r a n t i g e n i c t a r g e t molecules) are a l s o s u i t a b l e for u s e i n p a r a l l e l i m m u n o h i s t o c h e m i c a l s t u d i e s . T h e s e s t u d i e s c a n pro v i d e b o t h s p a t i a l a n d t e m p o r a l c o r r e l a t i o n of a d d u c t l o c a l i z a t i o n w i t h c e l l i n j u r y a n d t h u s p r o v i d e i n f o r m a t i o n o n p a t h o g e n e s i s t h a t c a n n o t be ob t a i n e d b y o t h e r m e a n s . T h e i n t e g r i t y of i n f o r m a t i o n g a i n e d f r o m i m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n of a n t i g e n s i s c r i t i c a l l y d e p e n d e n t o n k n o w l e d g e of t h e a n t i g e n i c d e t e r m i n a n t s t h a t t h e p r i m a r y a n t i b o d y w i l l bind. A s p a r t of t h e l o c a l i z a t i o n p r o c e d u r e , t h e a n t i g e n - b o u n d p r i m a r y a n t i b o d y i s d e t e c t e d b y a s y s t e m t h a t p e r m i t s d i r e c t o b s e r v a t i o n of t h e s i t e of a n t i b o d y b i n d i n g . A n u m b e r of d e t e c t i o n s y s t e m s are a v a i l a b l e t o enhance s e n s i t i v i t y a n d to visualize p r i m a r y antibody b o u n d i n s i t u to t a r g e t a n t i g e n . M o s t of t h e m u s e e i t h e r a r a d i o l a b e l e d s e c o n d a n t i b o d y a n d v i s u a l i z a t i o n b y a u t o r a d i o g r a p h y , or a n e n z y m e , fluorescent, or elec t r o n d e n s e p r o b e c o u p l e d t o a l i n k i n g s e c o n d a n t i b o d y , p r o t e i n A , or a v i d i n - b i o t i n a m p l i f i c a t i o n s y s t e m (1,2). I m m u n o h i s t o c h e m i s t r y p r o v i d e s s t r u c t u r a l s p e c i f i c i t y t h a t i s ab s e n t i n a u t o r a d i o g r a p h y of r a d i o l a b e l e d c o m p o u n d s a n d f u r t h e r affords t h e o p p o r t u n i t y t o s t u d y h u m a n t i s s u e w i t h o u t h a v i n g f i r s t to a d m i n i s t e r a r a d i o a c t i v e c o m p o u n d . I n a d d i t i o n , i m m u n o h i s t o c h e m i c a l tech n i q u e s h a v e t h e a d v a n t a g e t h a t m a n y a n t i g e n s are p r e s e r v e d b y s o l u t i o n s u s e d for h i s t o l o g i c f i x a t i o n , e.g., f o r m a l d e h y d e . T h u s , t i s s u e s p r e s e r v e d for o t h e r p u r p o s e s c a n be s t u d i e d r e t r o s p e c t i v e l y to c o r r e l a t e h i s t o p a t h o l o g y , a d d u c t l o c a l i z a t i o n a n d o t h e r i n d i c e s of i n j u r y . I n p r i n c i p a l , t h e m e t h o d l e n d s i t s e l f w e l l to i d e n t i f i c a t i o n of b i o m a r k e r s of t o x i c a n t e x p o s u r e i n t h e c o n t e x t of " m o l e c u l a r e p i d e m i o l o g y " . A l t h o u g h a v a r i e t y of f i x a t i o n t e c h n i q u e s are a v a i l a b l e , n o s i n g l e s y s t e m p r o v i d e s o p t i m a l p r e s e r v a t i o n of m o r p h o l o g y of a l l t i s s u e s a n d a l l t a r g e t a n t i g e n s s i m u l t a n e o u s l y . T h u s t h e r e i s a s p e c t r u m of effectiveness i n a c h i e v i n g g o o d p r e s e r v a t i o n of b o t h s t r u c t u r e a n d a n t i g e n i c i t y , i n c l u d i n g s e v e r a l h i g h l y s a t i s f a c t o r y t e c h n i q u e s (3-6). F o r p r o s p e c t i v e s t u d i e s , we h a v e s u c c e s s f u l l y p i o n e e r e d the s i m u l t a n e o u s p r e s e r v a t i o n of a d d u c t a n t i g e n i c i t y a n d t i s s u e s t r u c t u r e u s i n g f i x a t i o n b y m i c r o w a v e i r r a d i a t i o n (7). M i c r o w a v e i r r a d i a t i o n h a d been d e m o n s t r a t e d t o p r o v i d e s u p e r i o r p r e s e r v a t i o n of a n t i g e n s u s e d i n d i a g n o s t i c p a t h o l o g y . F o r 22 of 23 a n t i g e n s i n n i n e t i s s u e s i n c o m p a r i s o n w i t h formaldehyde, it yielded i m m u n o s t a i n i n g that was more intense and m o r e e x t e n s i v e w h i l e p r o v i d i n g e x c e l l e n t p r e s e r v a t i o n of c y t o m o r p h o l o g i c d e t a i l (8). B y c a u s i n g e x c i t a t i o n of w a t e r m o l e c u l e s , t h e m i c r o w a v e en e r g y effects c o n t r o l l e d h e a t i n g a n d c o a g u l a t i v e s t a b i l i z a t i o n of p r o t e i n .

Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

30.

BUCCI ET AL.

Antigenic Bio markers in Microwave-Fixed


Localization of D r u g - P r o t e i n A d d u c t s Prototype Hepatotoxin, Acetaminophen

in Animals

Target Tissues Dosed

with

329 the

I m m u n o l o g i c t e c h n i q u e s were u s e d t o s t u d y a c e t a m i n o p h e n t o x i c i t y i n m i c e t o e l u c i d a t e m e c h a n i s m s of l i v e r t o x i c i t y . T h e h e p a t o t o x i c i t y of acetaminophen is mediated b y a reactive metabolite, N-acetyl-p-benzoquinone i m i n e ( N A P Q I ) . T h e metabolite b i n d s to protein as 3 - ( c y s t e i n - S - y l ) a c e t a m i n o p h e n ( 3 - C y s - A ) a n d t h e a m o u n t of b i n d i n g corre lates w i t h t o x i c i t y . T h i s covalent b i n d i n g , a n d i n p a r t i c u l a r the 3 - C y s - A a d d u c t , i s t h e m o s t r e l i a b l e b i o m a r k e r of a c e t a m i n o p h e n t o x i c i t y (9-12). P r e v i o u s r e p o r t s d e s c r i b e d : a n E L I S A specific for 3 - C y s - A p r o t e i n a d d u c t s t h a t does n o t r e l y o n r a d i o l a b e l e d s a m p l e s (.13); c h a r a c t e r i z a t i o n of t h e r e l e v a n t epitope (14); a n d q u a n t i f i c a t i o n of 3 - C y s - A a d d u c t s i n l i v e r a n d s e r u m as a f u n c t i o n of h e p a t o t o x i c i t y (9). T h i s h i g h l y c h a r a c t e r i z e d a n t i s e r u m specific for t h e a c e t a m i n o p h e n - p r o t e i n a d d u c t , 3 - C y s - A , w a s u s e d t o c o r r e l a t e t h e i m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n of 3 - C y s - A pro t e i n a d d u c t s w i t h t h e d e v e l o p m e n t of c e l l i n j u r y , i n m i c r o w a v e - f i x e d l i v e r . A p p r o a c h . M a l e B 6 C 3 F 1 m i c e were f a s t e d o v e r n i g h t a n d d o s e d w i t h s a l i n e (control) or a c e t a m i n o p h e n (400 m g / k g ) i n t r a p e r i t o n e a l ^ t h e fol l o w i n g m o r n i n g . M i c e were k i l l e d at v a r i o u s t i m e s after d o s i n g a n d s e r u m a n d t i s s u e s were c o l l e c t e d for c l i n i c a l c h e m i s t r y a n d a d d u c t a n a l y s i s as d e s c r i b e d p r e v i o u s l y (9). T h e a l a n i n e a m i n o t r a n s f e r a s e ( A L T ) ac t i v i t y i n s e r u m w a s d e t e r m i n e d as a n i n d e x of h e p a t o t o x i c i t y (15). T o t a l h e p a t i c g l u t a t h i o n e ( G S H ) levels were d e t e r m i n e d , a c c o r d i n g t o t h e m e t h o d of T i e t z e (16), as a n i n d i c a t o r of t h e degree t o w h i c h r e a c t i v e m e t a b o l i t e h a d d e p l e t e d t h i s c o n s t i t u t i v e d e t o x i f i c a t i o n m e c h a n i s m (11). T h e p o l y c l o n a l a n t i s e r u m u s e d for t h e s e s t u d i e s w a s r a i s e d i n rab bits i m m u n i z e d w i t h 3-(N-acetylcystein-S-yl)acetaminophen-keyhole lim p e t h e m o c y a n i n a n d h a s s p e c i f i c i t y for a n t i g e n i c d e t e r m i n a n t s w h i c h are n o t f o u n d o n t h e p a r e n t c o m p o u n d or o n h o s t p r o t e i n , b u t are f o u n d o n a d d u c t s i n w h i c h a c e t a m i n o p h e n i s b o u n d v i a c a r b o n 3 t o t h e s u l f u r of c y s t e i n e i n p r o t e i n s (13,14, R o b e r t s et a l . , t h i s v o l u m e ) . C o m p a r i s o n of F i x a t i o n Techniques. F o r microwave fixation, fresh tis sues were t r i m m e d to a p p r o x i m a t e l y 2 m m t h i c k n e s s , p l a c e d i n p l a s t i c c a s s e t t e s , a n d h e l d i n ice c o l d s a l i n e (30-120 m i n u t e s ) u n t i l i r r a d i a t i o n i n a 7 0 0 - w a t t o v e n set to h o l d at 6 0 ° C for 2 m i n u t e s (7). M i c r o w a v e - f i x e d t i s s u e s were s t o r e d o v e r n i g h t i n 7 0 % e t h a n o l at r o o m t e m p e r a t u r e , proc essed r o u t i n e l y a n d then embedded i n paraffin, sectioned, deparaffinized, a n d r e h y d r a t e d u s i n g s t a n d a r d t e c h n i q u e s . O t h e r pieces of l i v e r were s i m i l a r l y t r i m m e d , then fixed b y i m m e r s i o n i n either 3.7% formaldehyde i n n e u t r a l p h o s p h a t e buffer (48 h o u r s ) , 2 % g l u t a r a l d e h y d e i n p h o s p h a t e buffer (2 h o u r s ) , B o u i n ' s s o l u t i o n (24 h o u r s ) , or B - 5 s o l u t i o n (4 h o u r s ) . T h e s e t i s s u e s were s i m i l a r l y e m b e d d e d i n p a r a f f i n t h e n s e c t i o n e d r o u t i n e l y . I n o t h e r s t u d i e s , s p e c i m e n s of l u n g a n d k i d n e y were f i x e d b y m i c r o w a v e i r r a d i a t i o n w i t h t h e s a m e s c h e d u l e as l i v e r .



330

T h e five f i x a t i o n p r o c e d u r e s were e v a l u a t e d s y s t e m a t i c a l l y to c o m p a r e p r e s e r v a t i o n of a d d u c t a n t i g e n i c i t y i n i m m u n o s t a i n e d s e c t i o n s a n d c e l l s t r u c t u r e i n h e m a t o x y l i n a n d e o s i n ( H & E ) - s t a i n e d s e c t i o n s ( T a b l e I). T a b l e I.

F i x a t i o n O u t c o m e : S i m u l t a n e o u s P r e s e r v a t i o n of A d d u c t Antigenicity and Tissue Structure

Structural Preservation

Antigen Preservation

Glutaraldehyde 2%

3*

3*

tissue brittle, i m m u n o s t a i n weak

B-5 solution

3

3.5

tissue brittle, i m m u n o s t a i n weak

Bounds solution

4

0

antigen not preserved

Formaldehyde 3.7%

4.5

4

immunostain and structure acceptable, s o m e cell shrinkage

Microwave irradiation

5

5

immunostain intense, uniform, boundaries sharp; structure excellent


Fixative

Comments

* s u b j e c t i v e scale 1-5; 5 = m a x i m u m score I m m u n o h i s t o c h e m i c a l S t a i n i n g . B a s e d o n t h e c o m p a r i s o n of f i x a t i o n t e c h n i q u e s , m i c r o w a v e i r r a d i a t i o n w a s u s e d for a l l s u b s e q u e n t e v a l u a t i o n s . A c e t a m i n o p h e n - p r o t e i n a d d u c t s were d e t e c t e d i n t i s s u e s e c t i o n s w i t h a m o d i f i c a t i o n of t h e u n l a b e l e d a n t i b o d y e n z y m e m e t h o d d e s c r i b e d b y S t e r n b e r g e r (1). D r u g - p r o t e i n a n t i g e n s were s t a i n e d b y s e q u e n t i a l i n c u b a t i o n s w i t h 1) r a b b i t a n t i 3 - C y s - A ( d i l u t e d 1:350 i n P B S c o n t a i n i n g 1% fetal b o v i n e s e r u m , 2 0 m i n u t e s ) , 2) sheep a n t i - r a b b i t l i n k i n g a n t i s e r u m , 2 0 m i n u t e s , 3) r a b b i t p e r o x i d a s e - a n t i p e r o x i d a s e ( P A P ) , 20 m i n u t e s , a n d 4) 3 , 3 ' - d i a m i n o b e n z i d i n e ( D A B ) s u b s t r a t e ( c o n t a i n i n g 0 . 0 3 % H2O2, 20 m i n u t e s ) . A l l i n c u b a t i o n s were at r o o m t e m p e r a t u r e a n d s l i d e s were w a s h e d t w i c e w i t h P B S b e t w e e n i n c u b a t i o n s . S e c t i o n s were c o u n t e r s t a i n e d w i t h M a y e r ' s h e m a t o x y l i n . T h e s e r e a g e n t s for i m m u n o h i s t o c h e m i c a l d e t e c t i o n of r a b b i t a n t i 3 - C y s - A were c o m p o n e n t s of a n immunoperoxidase staining k i t (Cambridge Research Laboratory, C a m b r i d g e , M A ) . T h e r e l a t i v e c o n t e n t of 3 - C y s - A p r o t e i n a d d u c t i n l i v e r c e l l s w a s d e t e r m i n e d b y t h e d e p o s i t i o n of t h e m i c r o s c o p i c a l l y v i s i b l e b r o w n D A B r e a c t i o n p r o d u c t a t the s i t e s of a n t i b o d y b i n d i n g . T h e


30.

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Antigenic Biomarkers in Microwave-Fixed

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a m o u n t of r e a c t i o n p r o d u c t w a s p r o p o r t i o n a l t o t h e q u a n t i t y of a d d u c t . T h e s u b j e c t i v e e s t i m a t e s of a d d u c t q u a n t i t y l o c a l i z e d i n h i s t o l o g i c sec t i o n s of l i v e r were c o r r o b o r a t e d b y q u a n t i t a t i v e i m m u n o a s s a y of a d d u c t i n l i v e r h o m o g e n a t e s ( R o b e r t s et a l . , t h i s v o l u m e ) . Correlation of A d d u c t Localization a n d T o x i c i t y . Saline-treated controls h a d no hepatocyte injury a n d no adduct formation. F i g u r e 1 is an H & E s t a i n e d s e c t i o n of l i v e r f r o m a s a l i n e - t r e a t e d m o u s e ( n o r m a l c o n t r o l ) a n d F i g u r e 2 i s a (negative) i m m u n o s t a i n for 3 - C y s - A a d d u c t , f r o m t h e s a m e animal. I n controls i n which normal pre-immune serum was substituted for a n t i 3 - C y s - A a n t i b o d y , n o i m m u n o h i s t o c h e m i c a l r e a c t i o n p r o d u c t w a s p r e s e n t i n s e c t i o n s f r o m a d d u c t - p o s i t i v e a n i m a l s ( F i g u r e 5). Acetaminophen-related hepatotoxicity was inferred from the mor p h o l o g i c c h a n g e s i n s e c t i o n s of l i v e r f i x e d b y m i c r o w a v e i r r a d i a t i o n , p r o c essed routinely, and stained w i t h H & E u s i n g standard techniques. F i g u r e 3 d e p i c t s a n H & E - s t a i n e d s e c t i o n of l i v e r f r o m a m o u s e k i l l e d t h r e e h o u r s after 4 0 0 m g / k g a c e t a m i n o p h e n . T h e c e n t r i l o b u l a r n e c r o s i s of a l l l o b u l e s w i t h s p a r i n g of p e r i p o r t a l areas i s c o n s i s t e n t w i t h p r e v i o u s d e s c r i p t i o n s of a c e t a m i n o p h e n - i n d u c e d h e p a t o t o x i c i t y a t t h e l i g h t m i c r o s c o p i c (10-11), a n d u l t r a s t r u c t u r a l (17) l e v e l s . T h r e e h o u r s after a dose of 4 0 0 m g / k g a c e t a m i n o p h e n t h e a r e a i m m u n o s t a i n e d for a d d u c t w i t h a n t i 3 - C y s - A a n t i b o d y ( F i g u r e 4), c o i n c i d e d e x a c t l y w i t h t h e n e c r o t i c zone e v i d e n t i n t h e H & E - s t a i n e d r e p l i c a t e sec t i o n f r o m t h e s a m e t i s s u e b l o c k ( F i g u r e 3). I n t i m e - c o u r s e s t u d i e s , t h e a d d u c t w a s p r e s e n t i n c e n t r i l o b u l a r h e p a t o c y t e s as e a r l y as 15 m i n u t e s after a d m i n i s t r a t i o n of a c e t a m i n o p h e n , w i t h p r o g r e s s i v e c o n c e n t r i c en l a r g e m e n t of t h e i m m u n o s t a i n e d area, w i t h i n c r e a s i n g i n t e n s i t y of s t a i n , t o a m a x i m u m a t t w o t o four h o u r s . T h e r e a f t e r , t h e i n t e n s i t y of s t a i n d e c r e a s e d i n a n i m a l s e x a m i n e d a t i n t e r v a l s t o 24 h o u r s , as h e p a t o c y t e s disintegrated. F i g u r e 6 is an i m m u n o s t a i n e d liver from a mouse killed s e v e n h o u r s after 4 0 0 m g / k g a c e t a m i n o p h e n . L e s s i n t e n s e a d d u c t s t a i n i n g w a s e v i d e n t , c o m p a r e d w i t h t h e 3-hour s a m p l e d e p i c t e d i n F i g u r e 4. T h e q u a n t i t y of a d d u c t i n h e p a t o c y t e s , as r e v e a l e d b y i m m u n o s t a i n i n g , c o r r e l a t e d w e l l w i t h t h e a d d u c t c o n t e n t of t h e 1 0 , 0 0 0 χ g l i v e r supernate from the same liver, measured b y quantitative i m m u n o a s s a y . F u r t h e r , i n dose-response s t u d i e s , a d d u c t a c c u m u l a t i o n i n h e p a t o c y t e s followed d e p l e t i o n of h e p a t o c e l l u l a r G S H . B y one h o u r after a 4 0 0 m g / k g dose, G S H w a s r e d u c e d b y > 9 0 % , a n d t h e a d d u c t w a s a b u n d a n t (7). W h e r e a s i m m u n o s t a i n i n g revealed a d d u c t as e a r l y as 15 m i n u t e s after a 4 0 0 m g / k g dose, m o r p h o l o g i c e v i d e n c e of h e p a t i c i n j u r y , as deter m i n e d b y l i g h t m i c r o s c o p y , w a s a b s e n t u n t i l one h o u r after d o s i n g . A t t h a t t i m e , c l o u d y s w e l l i n g of c e n t r i l o b u l a r h e p a t o c y t e s a n d " p i e c e - m e a l " n e c r o s i s of i n d i v i d u a l c e l l s were e v i d e n t . B y t w o t o t h r e e h o u r s , however, there was massive centrilobular necrosis, and significant hepatotoxicity w a s a l s o i n d i c a t e d b y a n i n c r e a s e i n A L T i n s e r u m (7). A v a i l a b l e evi dence i n d i c a t e s t h a t t h e 3 - C y s - A a d d u c t s t h a t appear i n s e r u m are




332

F i g u r e s 1-4. ( F i g u r e 1) N o r m a l l i v e r . S a l i n e - t r e a t e d c o n t r o l m o u s e . H & E s t a i n . C = c e n t r a l v e i n . ( F i g u r e 2) N o r m a l l i v e r . S a l i n e - t r e a t e d c o n t r o l m o u s e . A n t i 3 - C y s - A i m m u n o s t a i n for a c e t a m i n o p h e n a d d u c t i s n e g a t i v e . C = c e n t r a l v e i n . ( F i g u r e 3) M o u s e l i v e r 3 h o u r s after 4 0 0 m g / k g aceta minophen. H & E stain. Hepatocytes i n centrilobular region (within a r r o w s ) are s w o l l e n a n d v a c u o l a t e d . M a n y are a n u c l e a r . M o s t are nec rotic. T h e periportal region appears n o r m a l . C = central vein, Ρ = p o r t a l v e i n . ( F i g u r e 4) S a m e l i v e r as F i g u r e 3. A n t i 3 - C y s - A i m m u n o s t a i n for acetaminophen adduct is strongly positive i n centrilobular hepatocytes ( d a r k cells), a n d n e g a t i v e i n p e r i p o r t a l h e p a t o c y t e s . C = c e n t r a l v e i n , Ρ = portal vein.



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Antigenic Bio markers in Microwave-Fixed

Target Tissues

333

F i g u r e s 5-8. ( F i g u r e 5) S a m e l i v e r as F i g u r e 4. I m m u n o s t a i n c o n t r o l . P r e i m m u n e s e r u m w a s s u b s t i t u t e d for t h e o m i t t e d p r i m a r y a n t i b o d y t o v e r i f y t h e s p e c i f i c i t y of t h e i m m u n o s t a i n t o r e v e a l 3 - C y s - A . N o r e a c t i o n p r o d u c t i s p r e s e n t , d e s p i t e t h e presence of a d d u c t (compare w i t h F i g u r e 4). ( F i g u r e 6) M o u s e l i v e r 7 h o u r s after 4 0 0 m g / k g a c e t a m i n o p h e n . A n t i 3 - C y s - A i m m u n o s t a i n for a c e t a m i n o p h e n a d d u c t i s p o s i t i v e i n c e n t r i l o b u l a r h e p a t o c y t e s ( d a r k cells). A d d u c t h a s l e a c h e d f r o m l y s e d n e c r o t i c h e p a t o c y t e s n e a r e s t t h e c e n t r a l v e i n (C). T h e c e n t r i l o b u l a r h e p a t o c y t e s m o r e d i s t a l t o t h e c e n t r a l v e i n are a l s o n e c r o t i c , b u t s t i l l r e t a i n a d d u c t . S i n u s o i d s (arrows) are d i l a t e d . Ρ = p o r t a l v e i n . ( F i g u r e 7) M o u s e k i d n e y 3 h o u r s after 4 0 0 m g / k g a c e t a m i n o p h e n . A n t i 3 - C y s - A i m m u n o s t a i n for a c e t a m i n o p h e n a d d u c t i s p o s i t i v e ( d a r k cells) i n s o m e p r o x i m a l ( l o n g arrows) a n d some d i s t a l (short arrows) convoluted tubules. (Figure 8) M o u s e l u n g 3 h o u r s after 4 0 0 m g / k g a c e t a m i n o p h e n . A n t i 3 - C y s - A i m m u n o s t a i n for a c e t a m i n o p h e n a d d u c t i s p o s i t i v e i n m o s t e p i t h e l i a l c e l l s ( d a r k cells) l i n i n g t h e b r o n c h i o l e s . T h e r e m a i n d e r of t h e l u n g i s negative.



334


l e a c h e d f r o m d a m a g e d h e p a t o c y t e s (9). B y e i g h t h o u r s n e a r l y a l l c e n t r i l o b u l a r h e p a t o c y t e s were l y s e d , a n d t h e i n t e r v e n i n g s i n u s o i d s were d i l a t e d . T h e p r o g r e s s i v e l y s i s of n e c r o t i c c e l l s t h a t w a s e v i d e n t i n H & E s t a i n e d s e c t i o n s c o r r e l a t e d t e m p o r a l l y w i t h i n c r e a s e s i n s e r u m of b o t h A L T a n d a d d u c t s (data n o t s h o w n ) , l i b e r a t e d f r o m t h e d i s i n t e g r a t i n g h e p a t o c y t e s (9). A t 24 h o u r s , t h e r e w a s a m o d e r a t e i n f l a m m a t o r y re s p o n s e a t t h e j u n c t i o n of t h e n e c r o t i c c e n t r i l o b u l a r zone w i t h t h e s u r v i v i n g periportal hepatocytes, that included prominent phagocytosis b y m a c r o p h a g e s . T h e m a c r o p h a g e s a l s o c o n t a i n e d a d d u c t w h i c h w a s pre s u m a b l y scavenged from hepatocyte debris. B r i s k m i t o t i c a c t i v i t y and r e g e n e r a t i o n of h e p a t o c y t e s were a l s o p r e s e n t . T h e n e c r o t i c c e n t r i l o b u l a r r e g i o n c o l l a p s e d a n d w a s r e m o d e l e d over a t h r e e t o five-day p e r i o d . T h e u n i f o r m c e n t r i l o b u l a r d i s t r i b u t i o n of a d d u c t a n d of n e c r o s i s c o i n c i d e d w i t h t h e r e p o r t e d h e p a t i c d i s t r i b u t i o n of t h e e t h a n o l - i n d u c i b l e c y t o c h r o m e P - 4 5 0 e n z y m e p r e v i o u s l y s h o w n t o be t h e p r i m a r y e n z y m e r e s p o n s i b l e for c o n v e r s i o n of a c e t a m i n o p h e n to N A P Q I (18,19). T h e s e a d d u c t l o c a l i z a t i o n s t u d i e s f u r t h e r c o r r o b o r a t e t h e role of t h a t e n z y m e s y s t e m i n p r o d u c t i o n of t h e r e a c t i v e m e t a b o l i t e r e s p o n s i b l e for t h e ad duct formation. I n addition, the data support the concept t h a t N A P Q I r e a c t s w i t h p r o t e i n a t t h e s i t e of i t s f o r m a t i o n a n d does n o t diffuse f r o m c e n t r i l o b u l a r h e p a t o c y t e s t o o t h e r c e l l s s u c h as p e r i p o r t a l h e p a t o c y t e s . T h i s s y s t e m was also used to demonstrate 3 - C y s - A adducts i n the m o u s e k i d n e y a n d l u n g . T h r e e h o u r s after 4 0 0 m g / k g a c e t a m i n o p h e n , adduct was present i n m o s t p r o x i m a l a n d some d i s t a l convoluted tubules i n t h e k i d n e y ( F i g u r e 7). I n t h e l u n g , a d d u c t s were r e s t r i c t e d t o e p i t h e l i a l c e l l s l i n i n g t h e b r o n c h i a n d b r o n c h i o l e s , s i t e s p r e v i o u s l y r e p o r t e d t o be t h e t a r g e t of a c e t a m i n o p h e n - i n d u c e d n e c r o s i s (20) a n d t h e l o c a t i o n of t h e e t h a n o l - i n d u c i b l e P - 4 5 0 (19) ( F i g u r e 8). Conclusion U s i n g a d r u g - p r o t e i n a d d u c t as a b i o m a r k e r of c e l l i n j u r y , we h a v e s h o w n t h a t i m m u n o h i s t o c h e m i s t r y c a n be a p o t e n t t e c h n i q u e t o c o m p l e m e n t other i m m u n o c h e m i c a l analyses to reveal i n s i t u the molecular i n s u l t associated w i t h pathologic change at the subcellular level. W e l l c h a r a c t e r i z e d a n t i b o d y i s e s s e n t i a l , as are w e l l p r e s e r v e d t i s s u e s t r u c t u r e s a n d a d d u c t a n t i g e n i c i t y . C o r r o b o r a t i v e evidence afforded b y o t h e r t e c h n i q u e s , b o t h q u a n t i t a t i v e a n d t e m p o r a l , p r o v i d e s a m p l e v a l i d a t i o n of the localization procedure. I m m u n o h i s t o c h e m i s t r y is subjectively semi q u a n t i t a t i v e . C a r e f u l a t t e n t i o n t o p r o c e d u r a l d e t a i l s s u c h as s e c t i o n t h i c k n e s s , i n c u b a t i o n p e r i o d s , a n d s t a n d a r d i z e d m e t h o d s i m p r o v e repro ducibility and quantitation. A u t o m a t e d image analysis systems u s i n g d e n s i t o m e t r y or p h o t o m e t r y c a n provide further enhancement. These m e t h o d s c a n be e m p l o y e d u l t r a s t r u c t u r a l l y as w e l l , to p r o v i d e g r e a t e r d e t a i l r e g a r d i n g t h e l o c a l i z a t i o n of a n t i g e n i c m a r k e r s . O n c e v a l i d a t e d , i m m u n o h i s t o c h e m i c a l t e c h n i q u e s c a n be u s e d i n r e t r o s p e c t i v e s t u d i e s , t a k i n g a d v a n t a g e of t i s s u e s c o l l e c t e d for o t h e r p u r -



30. BUCCI ET AL

Antigenic Biomarkers in Microwave-Fixed Target Tissues

poses. For example, they can be used after the fact to identify possible toxic agents in human tissues. Immunohistochemical surveys provide a potential approach to environmental monitoring in which sentinel animals or plants could be collected and evaluated for presence of adducts that were biomarkers of exposure. Localization of a specific adduct in tissue provides a visual fixed point of reference from which further questions may be posed regarding other correlates of injury: altered structure or function and quantification of adduct. In the case of acetaminophen adducts in the liver, we showed that morphologic evidence of cell injury (histology), coincided consistently in time and location with quantity of localized adduct. Functional measurements revealed that clinical evidence of hepatic dysfunction also were proportional to histologic evidence of localized adduct and of injury. The concordance between adduct visualized in tissue sections and adduct measured in liver homogenates by quantitative immunoassay was temporally consistent as the adduct accumulated and also later as the adduct leaked from damaged cells and appeared in serum (7). Other correlations that strengthened the postulated mechanism of acetaminophen hepatotoxicity were the depletion of hepatic GSH antecedent to cell injury, and the coincident distribution of localized adduct, tissue injury, and ethanol-inducible P-450. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Sternberger, L.A. Immunocytochemistry; 3rd ed.; John Wiley: New York, 1986; Chapter 3. Rogers, A.W. Techniques of Autoradiography; Elsevier: Amsterdam, 1979. Falini, B. Arch. Pathol. Lab. Med. 1983, 107, 105-17. Pettigrew, N.M. Arch. Pathol. Lab. Med. 1989, 113, 641-4. Gerrits, P.O.; van Goor, H. J. Histotechnol. 1988, 11, 243-6. Rickert, R.R.; Maliniak, R.M. Arch. Pathol. Lab. Med. 1989, 113, 673-9. Roberts, D.W.; Hinson, J.A.; Benson, R.W.; Pumford, N.R.; Warbritton, A.R.; Crowell, J.A.; Bucci, T.J. Toxicologist 1989, 9, 47. Leong, A. S.Y.; Milie, J.; Duncis, C.G. J. Pathol. 1985, 156, 275-89. Pumford, N.R.; Hinson, J.A.; Potter, D.W.; Rowland, K.L.; Benson, R.W.; Roberts, D.W. J. Pharmacol. Exp. Ther. 1989, 248, 190-6. Mitchell, J.R.; Jollow, D.J.; Potter, W.Z.; Gillette, J.R.; Brodie, B.B. J. Pharmacol. Exp. Ther. 1973, 187, 211-7. Jollow, D.J.; Mitchell, J.R.; Potter, W.Z.; Davis, D.C.; Gillette, J.R.; Brodie, B.B. J. Pharmacol. Exp. Ther. 1973, 187 195-202. Gillette, J.R. Biochem. Pharmacol. 1974, 23, 2785-94. Roberts, D.W.; Pumford, N.R.; Potter, D.W.; Benson, R.W.; Hinson, J.A. J. Pharmacol. Exp. Ther. 1987, 241, 527-33.


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14. Potter, D.W.; Pumford, N.R.; Hinson, J.A.; Benson, R.W.; Roberts, D.W. J. Pharmacol. Exp. Ther. 1989, 248, 182-9. 15. Popper, H. In The Liver: Biology and Pathobiology; Arias, I.M., Jakoby, W.B., Popper, H., Schachter, D., and Shafritz, D.A., Eds., Raven Press, New York, 1988; p 1087-1103 16. Tietze, F. Anal. Biochem. 1969, 27, 502-22. 17. Placke, M.E.; Ginsberg, G.L.; Wyand, D.S.; Cohen, S. Toxicol. Pathol. 1987, 15, 431-8. 18. Anderson, L . M . ; Ward, J.M.; Park, S.S.; Rice, J.M. Path. Immunopathol. Res. 1989, 8, 61-94. 19. Okey, A.B. Pharmacol. Ther. 1990, 45, 241-298. 20. Jeffery, E.H.; Haschek, W.N. Toxicol. Appl. Pharmacol. 1988, 93, 452-61. RECEIVED August 30, 1990


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