Use of Urine for Monitoring Human Exposure to Genotoxic Agents

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Chapter 8

Use of Urine for Monitoring Human Exposure to Genotoxic Agents 1

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Richard H. C. San , Miriam P. Rosin , Raymond H. See , Bruce P. Dunn , and Hans F. Stich Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 2, 2018 | https://pubs.acs.org Publication Date: December 23, 1988 | doi: 10.1021/bk-1988-0382.ch008

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Environmental Carcinogenesis Unit, British Columbia Cancer Research Centre, 601 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada School of Kinesiology, Simon Fraser University, Burnaby, BC V5A 1S6, Canada We have recently assessed the feasibility of using two approaches for biological monitoring of pesticide-exposed individuals: (a) the detection of chromosome-damaging components in urine specimens, and (b) the analysis of urothelial cells for the presence of chromosomal damage, as measured by the frequency of micronuclei. Urine specimens were collected from non-smoking orchardists in the Okanagan Valley, B.C. during the pre-spraying and pesticide spraying periods of the fruit growing season. The urine was concentrated by preparative reversed-phase high-pressure liquid chromatography, and assayed for chromosome-damaging activity using Chinese hamster ovary cell cultures. A significant elevation in chromosome-damaging activity was observed only in the urine collected during the spraying period. Samples from the same individuals obtained during the pre-spraying period did not differ from non-smoking controls. Urothelial cells collected by centrifugation of urine from a single void proved to be insufficient in number for analysis of micronuclei. This approach may be applicable i f multiple urine samples are obtained. A brief description of our experience with micronuclei analysis in urothelial cells from individuals in other populations is also presented.

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Short-term in vitro assays of pure chemicals have aided in the dis­ covery of the genotoxic action of many chemicals. However, such tests do not permit the assessment of human exposure to environmental agents. The tests also do not simulate the number of complex ways in which chemical agents may interact in biological systems. At present, two methods are widely applicable for monitoring the effects of genotoxic chemicals on exposed populations. One method involves the analysis of in vitro target indicators such as microbial or mammalian cells for genotoxic damage after exposure to body fluids (e.g., urine) from exposed individuals. The other approach involves the analysis of cells and tissues of the exposed individual for evidence of cytogenetic damage such as micronuclei or chromosomal aberrations. c

0097-6156/89/0382-0098$06.00/0 1989 American Chemical Society

Wang et al.; Biological Monitoring for Pesticide Exposure ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 2, 2018 | https://pubs.acs.org Publication Date: December 23, 1988 | doi: 10.1021/bk-1988-0382.ch008

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The r a t i o n a l e b e h i n d the a n a l y s i s o f u r i n e i s based on the knowledge t h a t i n g e s t i o n o r m e t a b o l i c t r a n s f o r m a t i o n o f endogenous and exogenous s u b s t a n c e s may r e s u l t i n the e x c r e t i o n o f m e t a b o l i t e s i n t o t h e u r i n e (_1). The d e t e c t i o n o f the m e t a b o l i t e s i n u r i n e would i n d i c a t e t h a t a b s o r p t i o n o f the p r e c u r s o r compound has o c c u r r e d , and t h a t c e l l s i n v a r i o u s organs a r e a t r i s k o f exposure d u r i n g s y s t e m i c d i s t r i b u t i o n o f the p a r e n t compound. U n f o r t u n a t e l y , t h e concent r a t i o n o f g e n o t o x i c m e t a b o l i t e s i n the u r i n e may be r e l a t i v e l y low, r e q u i r i n g e x t r a c t i o n and c o n c e n t r a t i o n p r o c e d u r e s f o r adequate d e t e c t i o n . T h i s d i f f i c u l t y was a l l e v i a t e d by Yamasaki and Ames (2^) , who used n o n - p o l a r A m b e r l i t e XAD-2 r e s i n t o c o n c e n t r a t e compounds from the u r i n e o f c i g a r e t t e smokers. S i n c e t h e n , t h i s approach has been used t o examine many t y p e s o f exposure t h r o u g h l i f e s t y l e (_3) , d i e t ( 4 - 6 ) , o c c u p a t i o n (7-11), and m e d i c a l t r e a t m e n t (12,13). R e c e n t l y , p r e p a r a t i v e r e v e r s e d - p h a s e h i g h - p r e s s u r e l i q u i d chromat o g r a p h y was shown t o be s u p e r i o r t o XAD-2 r e s i n s i n terms o f p e r formance and r e c o v e r y i n the e x t r a c t i o n o f n o n - p o l a r u r i n a r y genot o x i c c o n s t i t u e n t s (14). The m i c r o n u c l e u s t e s t has been s u c c e s s f u l l y a p p l i e d t o e x f o l i a t e d u r i n a r y b l a d d e r c e l l s o f smokers and c o f f e e d r i n k e r s ( 1 5 ) , as w e l l as o f b i l h a r z i a l p a t i e n t s (16). M i c r o n u c l e i are a c e n t r i c chromosome fragments d e r i v e d from a b e r r a n t chromosomes (17). Their p r e s e n c e i n the c y t o p l a s m o f e x f o l i a t e d c e l l s as D N A - c o n t a i n i n g bodies i n d i c a t e s the c a p a c i t y of genotoxins t o i n f l i c t genetic damage on the d i v i d i n g b a s a l c e l l s o f the e p i t h e l i u m (15) . An i n c r e a s e i n the m i c r o n u c l e u s f r e q u e n c y o f the b l a d d e r c e l l s , t o g e t h e r w i t h a d e m o n s t r a t i o n o f g e n o t o x i c a c t i v i t y i n the u r i n e , would p r o v i d e s t r o n g e v i d e n c e o f exposure t o e n v i r o n m e n t a l c o n t a m i n a n t s . I n t h i s paper we r e p o r t on our s t u d y o f u r i n e g e n o t o x i c i t y and micronucleus frequency i n e x f o l i a t e d u r i n a r y bladder c e l l s of i n d i v i d u a l s o c c u p a t i o n a l l y exposed t o p e s t i c i d e s . Methodology Study Groups. In t h i s s t u d y , u r i n e samples were c o l l e c t e d from 21 o r c h a r d i s t s ( a l l non-smokers) i n the Okanagan V a l l e y , B.C., when they were engaged i n t h e a p p l i c a t i o n o f p e s t i c i d e s d u r i n g the f r u i t growing season. As c o n t r o l s , u r i n e was c o l l e c t e d from t h e s e same i n d i v i d u a l s d u r i n g the p r e - s p r a y i n g as w e l l as the p o s t - s p r a y i n g seasons. I n a d d i t i o n , 16 i n d i v i d u a l s from an a g r i c u l t u r a l r e s e a r c h s t a t i o n i n the Okanagan r e g i o n were r e c r u i t e d t o p r o v i d e u r i n e samples d u r i n g the same t i m e p e r i o d as t h e o r c h a r d i s t s . As c o n t r o l s o u t s i d e t h e f r u i t g r o w i n g r e g i o n , non-smoking i n d i v i d u a l s from Vancouver and Grand F o r k s , B.C. were r e c r u i t e d t o p r o v i d e one u r i n e specimen. C o l l e c t i o n of U r i n e Samples. I t was d e c i d e d t h a t a l o n g i t u d i n a l study would be most i d e a l f o r t h i s p r o j e c t s i n c e v a r i a b l e s such as m e t a b o l i c and l i f e s t y l e d i f f e r e n c e s would be e s s e n t i a l l y e l i m i n a t e d . A t l e a s t one s i n g l e u r i n e sample was c o l l e c t e d from each o f the o r c h a r d i s t s d u r i n g the peak p e s t i c i d e s p r a y i n g season. Initially, the specimen c o n s i s t e d o f a l l v o i d s c o l l e c t e d between 16 and 24 hours a f t e r p e s t i c i d e a p p l i c a t i o n . Based on the o b s e r v a t i o n s made on t h e s e samples, i t was s u s p e c t e d t h a t t h e c o l l e c t i o n o f u r i n e a t 16 t o 24 hours p o s t - s p r a y i n g may have passed the t i m e a t w h i c h p e s t i c i d e s

Wang et al.; Biological Monitoring for Pesticide Exposure ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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are m e t a b o l i z e d and e x c r e t e d . C o n s e q u e n t l y , a l l u r i n e specimens were c o l l e c t e d from the o r c h a r d i s t s w i t h i n t h e e n t i r e f i r s t 8 h o u r s a f t e r p e s t i c i d e a p p l i c a t i o n . A l a t e a f t e r n o o n sample was a l s o c o l l e c t e d from the same i n d i v i d u a l s d u r i n g t h e p r e - s p r a y i n g season t o s e r v e as a c o n t r o l . U r i n e samples from a l l c o n t r o l groups were c o l l e c t e d l a t e i n t h e a f t e r n o o n (4 t o 8 p.m.). A l l u r i n e specimens were c o l l e c t e d i n s t e r i l e 500-ml p o l y ­ e t h y l e n e b o t t l e s (Nalgene, R o c h e s t e r , NY) w i t h o u t any p r e s e r v a t i v e s . B o t t l e s were r e f r i g e r a t e d by t h e p a r t i c i p a n t s d u r i n g the c o l l e c t i o n period u n t i l f u l l . B e f o r e f r e e z i n g , each sample was c e n t r i f u g e d t o obtain e x f o l i a t e d urinary bladder c e l l s . The s u p e r n a t a n t was s t o r e d a t -20°C u n t i l a n a l y s i s . The volume, pH, and c r e a t i n i n e c o n t e n t o f each u r i n e sample were measured. The c r e a t i n i n e d e t e r m i n a t i o n , o b t a i n e d u s i n g a m o d i f i c a t i o n o f t h e p r o c e d u r e d e s c r i b e d by I o s e f s o h n (18), s e r v e d t o a d j u s t f o r v a r y i n g u r i n e c o n c e n t r a t i o n s and d i f f e r e n c e s i n body w e i g h t s between i n d i v i d u a l s . P r e p a r a t i o n o f C o n c e n t r a t e s from U r i n e Samples. U r i n e specimens were c o n c e n t r a t e d by r e v e r s e d - p h a s e h i g h - p r e s s u r e l i q u i d chromatography u s i n g a Waters Prep LC/System 500A p r e p a r a t i v e l i q u i d chromatograph (Waters S c i e n t i f i c , M i l f o r d , MA) and a Waters Prep Pak 500/C18 r e v e r s e d - p h a s e column. A l l t h e o r g a n i c m a t e r i a l on the column was e l u t e d w i t h 50% a c e t o n e / 5 0 % 2.5 niM p h o s p h o r i c a c i d (v/v) (4_) . The e l u a t e was r o t a r y e v a p o r a t e d a t 40°C t o remove t h e a c e t o n e , n e u t r a l ­ i z e d w i t h 1 M sodium h y d r o x i d e , and f r e e z e - d r i e d . A f t e r d r y i n g , the powdered u r i n e e x t r a c t was s t o r e d a t -20°C and r e d i s s o l v e d i n d o u b l e d i s t i l l e d water i m m e d i a t e l y b e f o r e use. Assay f o r C l a s t o g e n i c (Chromosome-Damaging) A c t i v i t y i n U r i n e . Urine e x t r a c t s were a s s a y e d f o r t h e i r c a p a c i t y t o induce chromosome and c h r o m a t i d damage i n c u l t u r e d C h i n e s e hamster o v a r y (CHO) fibroblasts. D i l u t i o n s o f t h e u r i n e e x t r a c t s were p r e p a r e d t o o b t a i n v a r i o u s c o n c e n t r a t i o n s o f c r e a t i n i n e e q u i v a l e n c e (14). Following addition o f t h e u r i n e e x t r a c t f o r 3 h o u r s , t h e CHO c e l l s were i n c u b a t e d f o r a f u r t h e r 16 h o u r s , t h e n a r r e s t e d a t metaphase w i t h c o l c h i c i n e f o r 3 h o u r s , h a r v e s t e d , s t a i n e d , and a n a l y z e d f o r the p r e s e n c e o f chromo­ some as w e l l as c h r o m a t i d b r e a k s and exchanges (19). Using t h i s e x p e r i m e n t a l p r o t o c o l , t h e c y t o g e n e t i c damage o b s e r v e d was p r e ­ d o m i n a n t l y (^96%) o f the c h r o m a t i d t y p e . Analysis of Micronuclei i n E x f o l i a t e d U r o t h e l i a l C e l l s . E x f o l i a t e d u r o t h e l i a l c e l l s were o b t a i n e d from f r e s h l y c o l l e c t e d u r i n e samples by c e n t r i f u g a t i o n , f i x e d i n e t h a n o l / g l a c i a l a c e t i c a c i d (3:1, v / v ) , dropped onto p r e c l e a n e d s l i d e s , a i r - d r i e d o v e r n i g h t , and s t a i n e d a c c o r d i n g t o t h e p r o c e d u r e s d e s c r i b e d by S t i c h e t a l . (20). A minimum o f 300 i n t a c t u r o t h e l i a l c e l l s were a n a l y z e d t o d e t e r m i n e the f r e q u e n c y o f m i c r o n u c l e a t e d c e l l s (20-22). S t a t i s t i c a l A n a l y s i s . A l l s t a t i s t i c a l comparisons were p e r f o r m e d u s i n g n o n - p a r a m e t r i c methods. The c l a s t o g e n i c a c t i v i t y o f u r i n e samples from t h e a g r i c u l t u r a l r e s e a r c h s t a t i o n workers b e f o r e and d u r i n g p e s t i c i d e exposure was e v a l u a t e d f o r s i g n i f i c a n c e u s i n g the Wilcoxon paired-sample t e s t . Tukey's m u l t i p l e comparisons t e s t was used t o e v a l u a t e d i f f e r e n c e s i n the c l a s t o g e n i c a c t i v i t y o f u r i n e from o r c h a r d i s t s d u r i n g d i f f e r e n t p e s t i c i d e exposure p e r i o d s . This t e s t was a l s o used t o examine d i f f e r e n c e s i n the mean c l a s t o g e n i c

Wang et al.; Biological Monitoring for Pesticide Exposure ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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a c t i v i t y among t h e t h r e e s t u d y groups ( o r c h a r d i s t s , a g r i c u l t u r a l r e s e a r c h s t a t i o n w o r k e r s , and r e f e r e n c e c o n t r o l s ) . The Mann-Whitney U t e s t was used t o e v a l u a t e u r i n a r y c r e a t i n i n e v a l u e s and u r i n a r y pH.

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Results C l a s t o g e n i c A c t i v i t y i n U r i n e from R e f e r e n c e C o n t r o l Group. The u r i n e o f t h e r e f e r e n c e c o n t r o l group e x h i b i t e d low b u t s i g n i f i c a n t l e v e l s o f c l a s t o g e n i c a c t i v i t y compared t o those found f o r c o n t r o l d i s t i l l e d water c o n c e n t r a t e s (P a

Group R e f e r e n c e c o n t r o l s (n=22) Research s t a t i o n personnel ( n = l l ) : Before spraying 5..4±2.0 During spraying 6..3±3.1 (P>0.05) O r c h a r d i s t s (n=22): 4..9±3.5 Before spraying During spraying: c d 19..9±10.2 (P0.50). I t t h e r e f o r e appears t h a t i n d i r e c t exposure t o p e s t i c i d e s may be i n s u f f i c i e n t t o induce any f u r t h e r chromosome-damaging a c t i v i t y i n t h e u r i n e above t h a t o f n o r m a l / b a s e l i n e l e v e l s . C l a s t o g e n i c A c t i v i t y i n U r i n e from O r c h a r d i s t s ( U r i n e C o l l e c t e d W i t h i n 16 t o 24 Hours o f P e s t i c i d e A p p l i c a t i o n ) . P e s t i c i d e exposure d i d n o t make t h e u r i n e more t o x i c towards t h e CHO c e l l s , s i n c e t h e r e f e r e n c e c o n t r o l u r i n e samples demonstrated t h e same e x t e n t o f toxicity. The frequency d i s t r i b u t i o n o f u r i n a r y c l a s t o g e n i c a c t i v i t y a t 4.0 mg/ml c r e a t i n i n e e q u i v a l e n c e d u r i n g b o t h t h e p r e - s p r a y i n g ( F i g u r e 1-A) and s p r a y i n g ( F i g u r e 1-C) p e r i o d s appears t o be s i m i l a r . D u r i n g these two time p e r i o d s , t h e u r i n e s o f about 90% o f t h e s u b j e c t s were unable t o i n d u c e more t h a n 5% a b e r r a n t metaphases. T h e r e f o r e p e s t i c i d e s p r a y i n g d i d n o t appear t o a f f e c t t h e u r i n a r y c l a s t o g e n i c a c t i v i t y o f t h e o r c h a r d i s t s . The maximum c l a s t o g e n i c a c t i v i t y o b t a i n e d o v e r t h e dose range o f 1.0 t o 8.0 mg/ml c r e a t i n i n e e q u i v a l e n c e i s p r e s e n t e d i n F i g u r e 2-A ( b e f o r e s p r a y i n g , and a t 16 t o 24 hours p o s t - s p r a y i n g ) . D e s p i t e i n t r a - and i n t e r - i n d i v i d u a l v a r i a t i o n s , t h e group mean v a l u e s o f c l a s t o g e n i c a c t i v i t y f o r t h e s e two s e t s o f samples were n o t s i g n i f i c a n t l y d i f f e r e n t (Table I ) .

Wang et al.; Biological Monitoring for Pesticide Exposure ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Subject: S2

A

105

B

Subject: S10

30-