Chapter 18
Immunoaffinity-Based Monitoring of Human Exposure to Aflatoxins in China and Gambia
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John D. Groopman and Audrey Zarba Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205
Monoclonal antibody immunoaffinity column chromatography (IAC) used in combination with high performance l i q u i d chromatography (HPLC) i s a useful technique that permits the concentration, resolution, quantification of aflatoxin B1 (AFB1), i t s metabolites, and the aflatoxin-DNA adducts from various body fluids and food samples of people chronically exposed to aflatoxin. Two population groups of men and women l i v i n g in Guangxi Province, People's Republic of China and the v i l l a g e of Keneba, The Gambia were studied and samples measured by combined IAC/HPLC. In both studies samples of blood, urine, and human milk were collected. The average daily intakes of AFB1 of men and women studied in Guangxi Province were 48.4 ug and 92.4 ug, respectively. Partial results of analyses of blood and urine for aflatoxin-DNA adducts are reported here. We found two advantages to using IAC/HPLC methods: (1) IAC/HPLC permits efficient analysis of many samples of body fluids and food or grain samples in a short time; (2) IAC/HPLC is non-destructive to the aflatoxin molecule so the same sample aliquot can be used for confirmatory analysis. Using epidemiological methods, the relationship between aflatoxin exposure and human l i v e r cancer has been hindered by inadequate data on aflatoxin consumption, excretion, metabolism, and the general poor quality of world-wide cancer morbidity and mortality s t a t i s t i c s . Molecular dosimetry methods are needed to help accurately assess an i n d i vidual s exposure to aflatoxins. This i s especially important because of the recent reclassification by the 1
0097-6156/91/0451-0207$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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208
IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS
I n t e r n a t i o n a l A g e n c y f o r R e s e a r c h on C a n c e r (IARC) o f a f l a t o x i n B l (AFB1) t o a C a t e g o r y I , known human c a r c i n o g e n (1). The d e v e l o p m e n t o f m o l e c u l a r d o s i m e t r y methods t o p e r m i t t h e m o n i t o r i n g o f an i n d i v i d u a l ' s a f l a t o x i n i n d u c e d g e n o t o x i c burden w i l l l e a d t o the i d e n t i f i c a t i o n of people at h i g h r i s k for developing disease long before c l i n i c a l manifestation occurs. I n t h i s c h a p t e r , we w i l l d e s c r i b e t h e u s e o f i m m u n o a f f i n i t y / H P L C t e c h n i q u e s f o r u r i n e , serum a l b u m i n , a n d f o o d a n a l y s i s and how t h i s method h a s b e e n a p p l i e d t o t h e b i o l o g i c a l m o n i t o r i n g o f people exposed t o a f l a t o x i n s i n C h i n a a n d The G a m b i a . Extensive l i t e r a t u r e reviews of the a f l a t o x i n f i e l d h a v e b e e n p u b l i s h e d and t h e r e a d e r s h o u l d r e f e r t o t h e f o l l o w i n g recent reviews that d e s c r i b e the t o x i c o l o g y (2); b i o l o g i c a l m o n i t o r i n g (3) and e p i d e m i o l o g i c a l a s p e c t s o f a f l a t o x i n s a n d l i v e r c a n c e r (4) f o r more c o m p l e t e d e s c r i p tions of research i n these areas. Immunoaffinity Aflatoxin
C h r o m a t o g r a p h y and M o l e c u l a r D o s i m e t r y
of
Urine T h e i n i t i a l p i l o t s t u d y was c o n d u c t e d i n G u a n g x i Province, People's Republic of China. This investigation e x p l o r e d b o t h t h e d i e t a r y consumption o f a f l a t o x i n B l and the u r i n a r y e x c r e t i o n of o x i d a t i v e a f l a t o x i n metabolites i n t h e same p e r s o n . The methods e m p l o y e d i m m u n o a f f i n i t y c h r o m a t o g r a p h y w i t h s u b s e q u e n t HPLC q u a n t i t a t i o n . T h e s e pharmacokinetic data are e s s e n t i a l i n determining the r e l a t i o n s h i p among a f l a t o x i n e x p o s u r e , b i o l o g i c a l l y e f f e c t i v e d o s e s , and l i v e r c a n c e r ( 5 - 8 ) . Twenty i n d i v i d u a l s were s e l e c t e d from a p r e s u m p t i v e h i g h a f l a t o x i n exposure group and u r i n e s a m p l e s were o b t a i n e d i n t h e m o r n i n g . The d a i l y i n t a k e o f AFB1 f r o m t h e d i e t , w h i c h i s p r i m a r i l y c o r n c o n t a m i n a t e d w i t h a f l a t o x i n B l , r a n g e d f r o m 13.4 t o 8 7 . 5 u g AFB1. U r i n e s a m p l e s f r o m f o u r i n d i v i d u a l s who h a d b e e n e x p o s e d t o t h e h i g h e s t l e v e l ( 8 7 . 5 ug) t h e p r e v i o u s d a y were p r e p a r e d w i t h t h e monoclonal a n t i b o d y i m m u n o a f f i n i t y column and t h e n i n d i v i d u a l a f l a t o x i n m e t a b o l i t e s were s e p a r a t e d b y a n a l y t i c a l HPLC. These prove the presence o f the major AFB1-DNA a d d u c t , A F B 1 - N 7 - G u a , and i n d i c a t e d t h a t t h e monoc l o n a l a n t i b o d y c o l u m n s , i n c o m b i n a t i o n w i t h HPLC, c a n q u a n t i f y a f l a t o x i n - D N A a d d u c t s i n human u r i n e s a m p l e s o b t a i n e d from i n d i v i d u a l s e n v i r o n m e n t a l l y exposed t o A F B 1 . The above e x p e r i e n c e s t i m u l a t e d a more e x t e n s i v e f o l low-up study i n Guangxi P r o v i n c e . To a s s e s s t h e dose and e x c r e t i o n o f AFB1 and i t s a d d u c t s i n c h r o n i c a l l y e x p o s e d p e o p l e , t h e f o l l o w i n g p r o t o c o l was d e v e l o p e d . The d i e t s o f 30 m a l e s and 12 f e m a l e s , a g e s r a n g i n g f r o m 25 t o 64 y e a r s , were s t u d i e d f o r one week and t h e t o t a l a f l a t o x i n i n t a k e was d e t e r m i n e d f o r e a c h d a y . U r i n e was o b t a i n e d i n two t w e l v e h o u r f r a c t i o n s f o r t h r e e c o n s e c u t i v e d a y s d u r i n g t h e one
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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18.
GROOPMAN AND ZARBA
Aflatoxins in China and Gambia
209
week c o l l e c t i o n p e r i o d . T h e s e u r i n e s a m p l e s were o b t a i n e d o n l y a f t e r d i e t a r y a f l a t o x i n l e v e l s had been measured f o r a t l e a s t t h r e e c o n s e c u t i v e d a y s and t h e u r i n e c o l l e c t i o n s were i n i t i a t e d on t h e f o u r t h d a y o f t h e p r o t o c o l . The a v e r a g e m a l e i n t a k e o f AFB1 was 4 8 . 4 ug p e r d a y a n d t h e mean t o t a l i n t a k e f o r t h e s e v e n d a y p e r i o d was 2 7 6 . 8 u g A F B 1 . T h e a v e r a g e f e m a l e d a i l y i n t a k e was 9 2 . 4 u g AFB1 p e r d a y . P r e p a r a t i v e i m m u n o a f f i n i t y c h r o m a t o g r a p h y was p e r f o r m e d on a l i q u o t s of the twelve hour u r i n e s . I n t o t a l , 256 u r i n e s a m p l e s were a n a l y z e d . Complete o x i d a t i v e a f l a t o x i n m e t a b o l i t e e x c r e t i o n i n t o u r i n e f o r e a c h t w e l v e h o u r s a m p l e p e r i o d was c a l c u l a t e d b y m u l t i p l y i n g t h e u r i n e v o l u m e by t h e c o n c e n t r a t i o n o f a f l a t o x i n s d e t e r m i n e d by r a d i o i m m u n o a s s a y i n t h e a l i q u o t o f u r i n e . F i g u r e 1 d e p i c t s a s c a t t e r p l o t comparison of a f l a t o x in intake with t o t a l oxidative aflatoxin metabolite excre tion. A l l t h e m a l e and f e m a l e d a t a were c o m b i n e d f o r t h i s presentation. The a f l a t o x i n i n t a k e d a t a r e p r e s e n t s t h e t o t a l i n t e g r a t e d i n g e s t i o n by an i n d i v i d u a l f o r t h e d a y b e f o r e u r i n e c o l l e c t i o n and d u r i n g t h e t h r e e d a y u r i n e collection. The e x c r e t i o n d a t a a r e t h e c o m p o s i t e o f a l l a f l a t o x i n metabolites excreted into the u r i n e during the t h r e e days o f u r i n e s a m p l i n g . These data r e v e a l t h a t d e s p i t e a twenty f o l d range o f a f l a t o x i n B l d i e t a r y i n t a k e the amount o f a f l a t o x i n e x c r e t e d g e n e r a l l y v a r i e d o n l y o v e r a three f o l d range. A l l u r i n e s a m p l e s were t h e n a n a l y z e d b y HPLC a n d AFM1, AFP1 and t h e m a j o r AFB-DNA a d d u c t s q u a n t i f i e d ( F i g u r e 2 ) . When t h e a f l a t o x i n DNA a d d u c t e x c r e t i o n l e v e l s i n u r i n e were c o r r e l a t e d t o a f l a t o x i n B l d i e t a r y i n t a k e amount i n u g p e r day, a dose dependent e x c r e t i o n i s seen. Because the a f l a t o x i n - D N A a d d u c t h a s a r e l a t i v e l y s h o r t h a l f l i f e i n DNA (2) t h i s dose dependent e x c r e t i o n p a t t e r n r e f l e c t s exposures d u r i n g t h e p r e v i o u s few d a y s . The c o r r e l a t i o n c o e f f i c i e n t f o r t h i s a s s o c i a t i o n o f a d d u c t e x c r e t i o n a n d AFB1 i n t a k e i s 0.6 w i t h a Ρ v a l u e o f l e s s t h a n 0.00001. Taken t o g e t h e r , it a p p e a r s t h a t t h e q u a n t i f y i n g o f a f l a t o x i n DNA a d d u c t s i n u r i n e i s a v a l i d compartment t o s a m p l e f o r a f l a t o x i n e x p o s u r e , b u t more d a t a must be c o l l e c t e d f o r d e v e l o p i n g a r i s k model f o r p e o p l e . Serum A l b u m i n A n o t h e r a p p l i c a t i o n o f t h e i m m u n o a f f i n i t y c h r o m a t o g r a p h y t e c h n i q u e was i n t h e a n a l y s i s o f AFB1 b o u n d t o serum a l b u m i n ( 9 ) . B l o o d s a m p l e s were c o l l e c t e d on Day 5 from t h e same i n d i v i d u a l s who p a r t i c i p a t e d i n t h e s t u d y d e s c r i b e d above. Serum a l b u m i n was s e l e c t i v e l y isolated from b l o o d and s u b j e c t e d t o e n z y m a t i c p r o t e o l y s i s using Pronase. A f l a t o x i n s p e c i f i c a d d u c t s were p u r i f i e d b y immu n o a f f i n i t y c h r o m a t o g r a p h y and q u a n t i f i e d b y c o m p e t i t i v e radioimmunoassay. A highly s i g n i f i c a n t c o r r e l a t i o n of a d d u c t l e v e l w i t h AFB1 i n t a k e ( r = 0 . 6 9 , Ρ l e s s t h a n 0.000001) was o b s e r v e d . From t h e s l o p e o f t h e r e g r e s s i o n
Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
210
IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS TOTAL MALE AND FEMALE DATA INTAKE COMPARED TO EXCRETION
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