Chapter 21
Immunological Detection and Quantitation of Carcinogen—DNA Adducts 1
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Mariko Tada , Misaki Kojima , Tomoyuki Shirai , Nobuyuki Ito , and Toshiteru Morita Downloaded via UNIV OF ARIZONA on July 28, 2018 at 18:15:08 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan First Department of Pathology, Nagoya City University Medical School, Mizuho-ku, Nagoya 467, Japan College of General Education, Osaka University, Toyonaka 560, Japan 3
Antibodies against DNA modified by 4-nitroquinoline -1-oxide(4NQO), and 3,2'-dimethyl-4-aminobiphenyl(DMAB) were raised in rabbits. On application in enzyme linked immunosorbent assays (ELISA), these antibodies proved highly specific for the DNA-adducts which were used as the immunogens or structurally related forms. Sensitive immunoassays for the detection of the adducts isolated from tissues of rats exposed to carcinogens could therefore be established using competitive ELISA. Levels as low as 5-7 fmol adducts in 5 ug of DNA i.e. 2-3 adducts per 107 nucleotides could be detected. Demonstration of adduct formation in rat organs by immunoperoxidase staining revealed that the adducts were found preferentially in target tissues after exposure to DMAB, the staining intensity increasing dose dependently. Indirect immunofluorescence staining of human fibroblast (NSF) cells treated with 4NQO also indicated adducts to be localized specifically in the nuclei after treatment with 0.25 uM at mean lethal dose. These assays should prove useful for elucidation of the levels, localization and persistence of carcinogen carcinogen-DNA adducts in target organs in vivo. The availability of methods for the direct measurement of specific DNA adducts should greatly facilitate studies on the biological significance of these lesions. In recent years, the immunoassay approach to detection of DNA damage has attracted interest because of the associated sensitivity and specificity, and the fact that DNA or other chemical adducts do not reqire radiolabeling. A large number of polyclonal and monoclonal antibodies have thus been elicited against carcinogen-DNA adducts(l-5) and used in highly sensitive immunoassays such as the enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) to quantitate adducts formed in DNA from animal tissues and cultured cells. With these assays, it has been possible to determine adduct formation in the fmol range. Moreover, immunohistochemical staining with these antibodies has allowed 0097-6156/91/0451-0246$06.00/0 © 1991 American Chemical Society Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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d e t e c t i o n o f the p r e c i s e l o c a l i z a t i o n and examination o f the k i n e t i c s of f o r m a t i o n and removal o f adducts a t both s i n g l e - c e l l and c e l l population l e v e l s . This i n f o r m a t i o n i s o f obvious s i g n i f i c a n c e f o r r i s k assessment and m o l e c u l a r epidemiology. Our own r e s e a r c h h a s c o n c e n t r a c t e d on ( i ) d e t a i l e d characterization of polyclonal antibodies against 4-nitroquinoline 1 - o x i d e ( 4 N Q 0 ) - m o d i f i e d DNA and 3 , 2 - d i m e t h y l - 4 - a m i n o b i p h e n y l ( D M A B ) - m o d i f i e d DNA, ( i i ) q u a n t i t a t i o n o f a d d u c t s i n b i o l o g i c a l s a m p l e s and ( i i i ) 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 o f t h e a d d u c t s i n t i s s u e s and c u l t u r e d c e l l s . 1
Experimental Methods Polyclonal Antibodies: Carcinogen m o d i f i e d DNA was p r e p a r e d a s d e s c r i b e d p r e v i o u s l y ( 6 , 7 ) . M o d i f i e d DNA samples used as immunogens c o n t a i n e d 18 pmol o f 4NQ0-adduct o r 11.0 pmol o f DMAB-adducts i n 1 ug of c a l f thymus DNA, t h i s i s 6 4NQ0 adducts o r 3.4 DMAB adducts p e r 10 n u c l e o t i d e s . The m o d i f i e d DNA (0.25-0.5mg) was e l e c t r o s t a t i c a l l y complexed t o e q u a l amounts o f m e t h y l a t e d b o v i n e serum a l b u m i n i n p h o s p h a t e b u f f e r e d s a l i n e ( P B S ) , and e m u l s i f i e d w i t h c o m p l e t e Freund s adjuvant. Immunization o f r a b b i t s and p u r i f i c a t i o n o f IgG f r a c t i o n s were performed as d e s c r i b e d p r e v i o u s l y ( 6 , 7 ) . Immunoassay (ELISA): M i c r o t i t e r w e l l s (Nunc Type I I ) coated w i t h lOng o f h i g h l y m o d i f i e d DNA(l-6 adducts/10 n u c l e o t i d e s ) were used f o r c o m p e t i t i v e ELISA. A n t i b o d i e s were d i l u t e d i n PBS c o n t a i n i n g 2 % FCS and mixed w i t h e q u a l volumes o f s e r i a l l y d i l u t e d c o m p e t i t o r . The m i x t u r e s were i n c u b a t e d i n t h e w e l l s f o r 60 min a t 37°C, and t h e p l a t e s were then washed and i n c u b a t e d w i t h peroxidase-conjugated g o a t a n t i - r a b b i t I g G f o r one h o u r a t 37°C. Enzyme a s s a y s were c a r r i e d out a s d e s c r i b e d p r e v i o u s l y ( 6 ) . The amount o f i n h i b i t o r r e q u i r e d f o r 50 % i n h i b i t i o n o f a n t i b o d y b i n d i n g t o i m m o b i l i z e d a n t i g e n ( I A ^ Q ) was regarded as a measure o f t h e c o m p e t i t i v e ELISA sensitivity. (Figure 1.) !
Immunohistochemical
Staining
Rat T i s s u e s : 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 was c a r r i e d o u t a s described p r e v i o u s l y ( 1 4 ) . B r i e f l y , s e c t i o n s were t r e a t e d w i t h RNase A(100 ug/ml) a t 37°C f o r 2 h r and then w i t h 2.5N HC1 a t room temperature f o r 45 min. S l i d e s were i n c u b a t e d w i t h 5 % normal goat serum f o r 20 min and then w i t h anti-DMAB-DNA a n t i b o d y f o r 60 min a t 37°C. A f t e r treatment w i t h 0.3 % H 0 ? f o r 30 min a t 37°C, b i n d i n g o f the a n t i b o d y was v i s u a l i z e d by the ABC method(37°C f o r 30 min) w i t h 0.05 % d i a m i n o b e n z i d i n e ( f o r 10 m i n ) . The s e c t i o n s were a l s o weakly counterstained w i t h hematoxylin b e f o r e d e h y d r a t i o n through graded e t h a n o l s and x y l e n e , and mounting. Human C e l l s : Normal human s k i n f i b r o b l a s t c e l l s (NHSF) were grown as monolayer c u l t u r e s on Lab-Tek 2 c h a m b e r - s l i d e s (Lab-Tek P r o d u c t s , N a p e r v i l l e , 1 1 1 ) and t r e a t e d w i t h 2§10 χ 1 0 M 4NQ0 i n Hank's solution. T h e s e c e l l s a m p l e s were a p p l i e d t o i m m u n o f l u o r e s e n c e s t a i n i n g a c c o r d i n g t o our method ( 6 ) . 2
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_i_ 10
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f n o l DMAB-adduct
F i g . 1 Competitive i n h i b i t i o n of antibody b i n d i n g t o DMAB-DNA by ELISA. Each w e l l was coated w i t h 10 ng o f d e n a t u r a t e d DMAB-DNA (5 pmol adducts/ug DNA) and a n t i b o d y was 25 ng o f IgG p e r w e l l . The i n h i b i t e r c o n t a i n e d fmol a d d u c t s i n ug o f DNA ; ( · ) 26, ( O ) 710, ( Δ ) 5,140 and ( • ) 13,600, r e s p e c t i v e l y .
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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Results
Characterization of Antibodies j_ Anti 4NQ0-DNΑ-Antibody: The a n t i b o d y a f f i n i t y f o r 4NQ0-DNA was s t u d i e d i n d e t a i l by c o m p e t i t i v e ELISA u s i n g DNA w i t h l a r g e d i f f e r e n c e s i n m o d i f i c a t i o n l e v e l s . The r e s u l t s p r e s e n t e d i n T a b l e I , c l e a r l y show t h a t h i g h l y m o d i f i e d DNA i s r e c o g n i z e d more e f f i c i e n t l y by t h e a n t i b o d y than DNA h a v i n g a lower d e g r e e o f m o d i f i c a t i o n . F o r measurement o f a d d u c t s i n DNA f r o m b i o l o g i c a l s a m p l e s , s t a n d a r d m o d i f i e d DNA s h o u l d be u s e d a s t h e c o m p e t i t o r i n t h e same m o d i f i c a t i o n r a n g e a s t h e s a m p l e s ( 1 - 1 0 adducts/10 n u c l e o t i d e s ) . As t h e anti-4NQ0-DNA a n t i b o d y h a r d l y r e a c t e d w i t h n u c l e a s e P i d i g e s t e d n u c l e o t i d e a d d u c t s , i t may n o t i n f a c t r e c o g n i z e d t h e 4NQ0 a d d u c t m o i e t y i n DNA, b u t r a t h e r may r e a c t w i t h s u r r o u n d i n g DNA s t r u c t u r e s . The a n t i b o d y was a l s o found t o r e c o g n i z e DNA m o d i f i e d by 4HAQ0 d e r i v a t i v e s such as 6 - m e t h y l - 4 - n i t r o q u i n o l i n e 1-oxide ( 6 - C H 3 4NQ0) o r N - m e t h y l - 4 - n i t r o q u i n o l i n e 1-oxide ( N - C H 3 - 4 N Q O ) w i t h t h e same a f f i n i t y as 4NQ0-DNA. The adducts r e c o g n i z e d by t h e a n t i b o d y a r e presumably heat and a l k a l i n e s t a b l e , s i n c e t h e r e a c t i v i t y o f 4NQ0-DNA to a n t i b o d i e s d i d n o t decrease a f t e r heat treatment i n PBS o r 0.1N NaOH. Table I . Competitive i n h i b i t i o n of p o l y c l o n a l antibody b i n d i n g t o 4NQ0 DNA Modification adducts fmol/ug DNA
Competitor
s s 4NQ0-DNA
a)
ds 4NQ0-DNA
ss ss ss ss
AFF-DNA DMAB-DNA 6CH -4NQ0-DNA N-CH -4NQ0-DNA 3
3
20,000 1,860 356 36.3 21.0 10.0 20,000 1,860 356 25,000 13,600 13,630 12,360
Amount o f c o m p e t i t o r (fmol) causing 50% i n h i b i t i o n o f antibody binding 5 15 60 120 120 120 20
>iof) 5
>10
>10 5 5
5
The m i c r o t i t e r p l a t e s were coated w i t h 50 ng o f ds 4NQ0-DNA (20 pmol adducts/ug) and 25 ng o f IgG were a p p l i e d per w e l l . a) M o d i f i e d DNAs were heated i n PBS o r 0.1N NaOH a t 90°C f o r 4 min. b) No i n h i b i t i o n d e t e c t e d a t t h e h i g h e s t c o n c e n t r a t i o n tested. A n t i DMAB-DNA A n t i b o d y : S p e c i f i c f o r DMAB-DNA a n d 4a m i n o b i p h e n y l ( A B ) - m o d i f i e d DNA, t h i s a n t i b o d y was found t o n o t c r o s s r e a c t w i t h 4NQ0-,4-aminoazoben d e r i v a t i v e - o r AAF-modified DNA ( T a b l e I I ) . I t i s t h e r e f o r e c o n c l u d e d t h a t t h e anti-DMAB-DNA a n t i b o d y r e c o g n i z e s b i p h e n y l r i n g s bound t o p u r i n e bases ( 7 ) .
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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I n c o n t r a s t t o anti-4NQ0-DNA a n t i b o d y , t h e I A ^ Q v a l u e s f o r heated denatured DNA were 7-8 f m o l / a s s a y , independent o f m o d i f i c a t i o n l e v e l s and almost same a f f i n i t y f o r enzymatic h y d r o l y s a t e s (mono- o r o i l i g o n u c l e o t i d e s ) . DMAB n u c l e o t i d e s a r e thought t o be t h e a n t i g e n i c determinants. (Table I I ) The a n t i b o d y has a g r e a t e r a f f i n i t y f o r h i g h l y m o d i f i e d n a t i v e DMAB-DNA than f o r low m o d i f i e d DNA. D e n a t u r a t i o n o f DNA proved e s s e n t i a l f o r c o m p e t i t i v e ELISA. Table I I .
C o m p e t i t i v e i n h i b i t i o n o f a n t i b o d y b i n d i n g t o DMABDNA by v a r i o u s c o m p e t i t o r s
Competitor
s s DMAB-DNA
a)
ds DMAB-DNA
b)
DMAB-DNA-digest ss AB-DNA ' s s 4NQ0-DNA ss AAF-DNA ss 2-MeO-AAB-DNA ss 3-MeO-AAB-DNA C
Modification (fmol/ug)
Amount o f c o m p e t i t o r
13,600 710 26 13,600 710 26 13,600 866 20,000 25,000 15,600 11,300
7.0 7.7 8.0 22.0 44.0 90.0 30.0 >10f ) 5
>10 _
>10^ >10 5
The m i c r o t i t e r p l a t e s were coated w i t h 50 ng o f ds DMAB-DNA (12.3 pmol/ug) and 20 ng of IgG were a p p l i e d per w e l l . a) Heated i n PBS o r 0.1 Ν NaOH a t 90°C f o r 5 min. b) DNasel d i g e s t e d . c) 4-Aminobiphenyl adducts were determined s p e c t r o p h o t o m e t r i c a l l y . d) No i n h i b i t i o n d e t e c t e d a t the h i g h e s t c o n c e n t r a t i o n t e s t e d . Quantitation of Adducts i n DNA from Rat Tissues 4NQO: D o n r y u f e m a l e r a t s ( 8 weeks o l d ) were a d m i n i s t e r e d an i n t r a v e n o u s i n j e c t i o n o f [ H]4HAQ0 a t 20 mg p e r k g body w e i g h t . A f t e r one hour, the l i v e r , pancreas, k i d n e y , l u n g and u t e r u s t i s s u e s were e x c i s e d and DNA samples i s o l a t e d by phenol e x t r a c t i o n combined w i t h p r o t e a s e and RNase A t r e a t m e n t . The amounts o f 4NQ0 adducts were determined by UV absorbance a t 260 nm and by measurement o f radioactivity. The DNA samples were denatured by h e a t i n g f o r 5 min a t 90°C, and were assayed by c o m p e t i t i v e ELISA. C o m p e t i t i v e ELISA was c a r r i e d o u t u s i n g h i g h l y m o d i f i e d DNA (107 a d d u c t s / 1 0 n u c l e o t i d e s ) and s l i g h t l y m o d i f i e d DNA ( 1 - 3 adducts/10 n u c l e o t i d e s ) as standards. The r e s u l t s a r e shown i n T a b l e I I I and compared w i t h v a l u e s o b t a i n e d from t h e r a d i o a c t i v i t y measurements. Using the s l i g h t l y modified DNA ( 3 adducts/10 n u c l e o t i d e s ) f o r the standard i n h i b i t i o n curve, the values obtained w e r e e s s e n t i a l l y t h e same a s t h o s e g a i n e d f r o m radioactivity m e a s u r e m e n t s . U s i n g t h e h i g h l y m o d i f i e d DNA ( 1 0 a d d u c t s / 1 0 n u c l e o t i d e s ) as the s t a n d a r d , the v a l u e s were a p p r o x i m a t e l y 30% lower than r a d i o a c t i v i t y assay l e v e l s . The pancreas has been found t o be the main t a r g e t organ a f t e r a s i n g l e i . v i n j e c t i o n o f 4HAQ0, t h e 6
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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h i g h e s t amounts o f adducts were a l s o found i n p a n c r e a t i c t i s s u e . Adducts were not p r e s e n t o r o n l y p r e s e n t a t low l e v e l s i n t h e l i v e r n o n t a r g e t organ ( 9 ) . These r e s u l t s i n d i c a t e a s i g n i f i c a n t r o l e o f adducts f o r m a t i o n i n t u m o r i g e n e s i s . T a b l e I I I . Q u a n t i t a t i o n o f 4NQ0-adducts i n DNA from r a t t i s s u e s by r a d i o a c t i v i t y and c o m p e t i t i o n ELISA J
Tissue
Amount o f [ H ] 4HAQ0 (fmol/ug DNA) ELISA
Radioactivity
Pancreas Kidney Stomach Uterus Lung Liver
392.0 232.0 221.0 216.7 87.6 4.0
(a)
(b)
373.3 220.0 206.7 208.3 80.6
