Biological Markers To Study Exposure in Animals and Bioavailability

Chapter 7

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Biological Markers To Study Exposure in Animals and Bioavailability of Environmental Contaminants L. R. Shugart, S. M . Adams, B. D. Jiminez, S. S. Talmage, and J. F. McCarthy Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 Epidemiologic studies of agents present in the environment seek to identify the extent to which they contribute to the causation of a specific toxic, clinical, or pathological endpoint. The multifactorial nature of disease etiology, long latency periods and the complexity of exposure, a l l contribute to the difficulty of establishing associations and causal relationships between a specific exposure and an adverse outcome. These barriers to studies of exposure and subsequent risk assessment cannot generally be changed. However, the appropriate use of biological markers in animal species living in a contaminated habitat can provide a measure of potential damage from that exposure and, in some instances, act as a surrogate for human environmental exposures. Quantitative predictivity of the effect of exposure to environmental pollutants is being approached by employing an appropriate array of biological end points. Human exposure to environmental pollutants is often exceedingly complex. Sources of exposure are multiple, and the importance of a particular source may depend on individual practices. Humans are highly mobile, and direct study, particularly when physiological testing is required, is generally expensive and logistically difficult. For these reasons, a simpler animal model which provides a parallel biological marker in humans would be useful. Small mammals have frequently been used to monitor the presence of metals and other contaminants in the environment. Such biomonitoring can be essential for determining bioavailability and resultant biological effects under natural conditions. Small mammals can be particularly effective biomonitors of soil contaminants i f the species used are abundant, easily caught, do not migrate long distances, and have well documented food habits This chapter not subject to U.S. copyright Published 1989 American Chemical Society

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


86

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

(1). Good b i o m o n i t o r s a r e i n e q u i l i b r i u m w i t h the environment and s h o u l d show a graded response t o a range o f p o l l u t a n t s . Data a r e s p a r s e on the amounts o f hazardous s u b s t a n c e s r e l e a s e d to the environment. M e a s u r i n g c o n c e n t r a t i o n s o f t h e s e s u b s t a n c e s i n s o i l , p l a n t s , and a n i m a l s would r e q u i r e an e x h a u s t i v e m o n i t o r i n g program w h i c h would be c o s t l y , e s p e c i a l l y s i n c e many c h e m i c a l s w i l l n o t be p r e s e n t as the p a r e n t compound i n l i v i n g organisms due t o t h e i r r a p i d metabolism. The i m p o r t a n t q u e s t i o n i s n o t whether t h e s e s u b s t a n c e s a r e p r e s e n t , b u t r a t h e r whether t h e s e s u b s t a n c e s e n t e r the b i o t a i n c h e m i c a l forms and c o n c e n t r a t i o n s t h a t w i l l r e s u l t i n an a d v e r s e b i o l o g i c a l e f f e c t . Thus, m o n i t o r i n g the b i o l o g i c a l a v a i l a b i l i t y o f t h e s e s u b s t a n c e s i n b i o t a becomes a c r i t i c a l parameter f o r d e t e r m i n i n g whether t h e y have p o t e n t i a l f o r b e i n g hazardous and whether t h e y c o u l d r e s u l t i n s i g n i f i c a n t exposures t o humans ( 2 ) . The e s t i m a t i o n o f the r i s k r e s u l t i n g from exposure t o e n v i r o n m e n t a l p o l l u t a n t s r e q u i r e s knowledge o f the dose and p h a r m a c o k i n e t i c s o f the c h e m i c a l s i n the a n i m a l . A s y s t e m i c dose i n an i n d i v i d u a l can be o b t a i n e d by q u a n t i t a t i o n o f the c h e m i c a l o r i t s m e t a b o l i t e s i n body f l u i d s such as b l o o d , u r i n e , f e c e s , sputum, or m i l k , however, a major drawback t o t h i s approach i s t h a t the a n a l y s i s must be performed s h o r t l y a f t e r exposure, t h a t i s , p r i o r to the c l e a r a n c e o f the m e t a b o l i t e s from the body. A l t e r n a t i v e p r o c e d u r e s f o r m o n i t o r i n g exposure t o hazardous s u b s t a n c e s a r e needed. I n p a r t i c u l a r , new methods a r e needed f o r measuring r e a c t i o n p r o d u c t s o f c h e m i c a l s , such as m e t a b o l i t e s and a d d u c t s , i n a n i m a l s and humans t h a t c o r r e l a t e w i t h p a t t e r n s o f a s s o c i a t e d b i o l o g i c a l change and p a t h o l o g i c a l c o n d i t i o n s Q , 4 ) . Our approach t o d e t e c t i n g exposure t o hazardous c h e m i c a l s i n the environment, and t o e s t i m a t i n g the p o t e n t i a l f o r subsequent adverse e f f e c t s , i s to monitor b i o l o g i c a l endpoints i n w i l d a n i m a l s . B i o l o g i c a l e n d p o i n t s i n c l u d e i n d i c a t o r s t h a t : (a) q u a n t i f y exposure by measuring c h e m i c a l i n t e r a c t i o n w i t h c r i t i c a l m o l e c u l a r t a r g e t s (adducts i n n u c l e i c a c i d s and p r o t e i n s ) ; (b) measure a l t e r a t i o n s i n s p e c i f i c , s e n s i t i v e , and c r i t i c a l p h y s i o l o g i c a l and b i o c h e m i c a l p r o c e s s e s ( d e t o x i f y i n g enzyme a c t i v i t i e s and m e t a l - b i n d i n g p r o t e i n s ) ; and, ( c ) m o n i t o r e a r l y e x p r e s s i o n o f c e l l u l a r d y s f u n c t i o n ( n e o p l a s i a o r l o s s o f immune competence). U s i n g t h i s approach the organism i s seen t o f u n c t i o n as an i n t e g r a t o r o f exposure, a c c o u n t i n g f o r a b i o t i c and p h y s i o l o g i c a l f a c t o r s t h a t modulate the dose o f t o x i c a n t t a k e n up from the environment. The b i o l o g i c a l marker i s used t o d e t e c t the response o f the organism t o the e n v i r o n m e n t a l i n s u l t . Because o f the o f t e n l o n g l a t e n t p e r i o d between exposure and subsequent i r r e v e r s i b l e d e l e t e r i o u s impact on h e a l t h , i t would be d e s i r a b l e t o have s e n s i t i v e b i o l o g i c a l i n d i c a t o r s t h a t c o u l d d e t e c t exposure e a r l y enough so t h a t p r e v e n t i v e o r r e m e d i a l measures c o u l d be t a k e n . Three elements a r e c r i t i c a l t o our approach: (a) marker s e l e c t i o n based on t o x i c o l o g i c a l mechanisms; (b) f i e l d s t u d i e s t o e s t a b l i s h c o r r e l a t i o n s between e n v i r o n m e n t a l c o n t a m i n a t i o n and markers; and, ( c ) l a b o r a t o r y c o n f i r m a t i o n o f c a u s a l r e l a t i o n s h i p s between exposure, b i o l o g i c a l markers, and adverse e f f e c t s . B i o l o g i c a l m o n i t o r i n g can be used as a c o s t - e f f e c t i v e s u r v e y o f c o n t a m i n a t e d s i t e s , f o r r o u t i n e m o n i t o r i n g o f uncontaminated s i t e s ,


7.

SHUGART ET AL.

87

Biological Markers in Animals


t o p r o v i d e e v i d e n c e f o r contaminant impact on b i o t a , and t o e s t a b l i s h p r i o r i t i e s f o r s i t e cleanup. A s u i t e o f b i o m a r k e r s t h a t we c u r r e n t l y u s e t o m o n i t o r f o r exposure t o s e l e c t e d e n v i r o n m e n t a l p o l l u t a n t s , i s shown i n T a b l e I . I n f o r m a t i o n o b t a i n e d upon m o n i t o r i n g t h e response o f t h e s e b i o m a r k e r s i s compared t o l o n g - t e r m adverse e f f e c t s i n t h e organism (reduced f e c u n d i t y , d e c r e a s e d d i s e a s e r e s i s t a n c e , and tumor formation). T a b l e I . B i o l o g i c a l Markers o f Exposure and B i o a v a i l a b i l i t y o f E n v i r o n m e n t a l Contaminants

Environmental Pollutant

A.

B.

Toxic Metals: Cu, Hg, Ag, Zn, Cd, Pb,

O r g a n i c Cmpds: 1. PAH's

Reliability Index*

Biological Marker

DNA i n t e g r i t y / m d C y d metal-binding proteins porphyrin biosynthesis immune response x e n o b i o t i c metabolism

s s,d s,d,p s s

DNA/hemoglobin adducts x e n o b i o t i c metabolism immune response DNA i n t e g r i t y / m d C y d

s, d,p s,d s s

x e n o b i o t i c metabolism porphyrin p r o f i l e immune response DNA i n t e g r i t y / m d C y d DNA/hemoglobin adducts

s s s s s,d

5

5

2.

PCB's & TCDD

5

*

s: s i g n a l o f p o t e n t i a l problem d: d e f i n i t i v e i n d i c a t o r o f type o r c l a s s o f p o l l u t a n t p: p r e d i c t i v e i n d i c a t o r o f l o n g - t e r m adverse e f f e c t

S t u d i e s w i t h C e l l u l a r Biomarkers DNA M o d i f i c a t i o n . As a r e s u l t o f exposure t o hazardous s u b s t a n c e s i n t h e environment, i t i s n o t u n r e a s o n a b l e t o e x p e c t t h a t damage t o DNA may o c c u r t h a t goes u n c o r r e c t e d , w i t h subsequent a d v e r s e e f f e c t s t o t h e organism. The r a t i o n a l e u n d e r l y i n g t h e s t r a t e g y o f c h e m i c a l d o s i m e t r y by d e t e r m i n i n g l e v e l s o f compounds which become c o v a l e n t l y bound t o c e l l u l a r macromolecules i s based on o u r u n d e r s t a n d i n g o f t h e mode o f a c t i o n o f g e n o t o x i c compounds (5-7) . The m e t a b o l i s m o f c h e m i c a l s i s a r e l a t i v e l y complex phenomenon and d e t o x i f i c a t i o n by c e l l u l a r mechanisms i s n o t always complete, sometimes r e s u l t i n g i n h i g h l y r e a c t i v e e l e c t r o p h i l i c m e t a b o l i t e s . These m e t a b o l i t e s c a n



88


undergo a t t a c k by n u c l e o p h i l i c c e n t e r s i n macromolecules such as l i p i d s , p r o t e i n s , DNA and RNA which o f t e n r e s u l t s i n c e l l u l a r toxicity. B i n d i n g w i t h DNA, however, can cause f o r m a t i o n o f a l t e r e d bases t h a t can be r e p a i r e d , be innocuous, or r e s u l t i n a l t e r a t i o n s w h i c h become f i x e d and are t r a n s m i t t e d t o daughter cells. C u r r e n t r e s e a r c h suggests t h a t r e a c t i o n o f c h e m i c a l s w i t h DNA and the e n s u i n g changes w h i c h r e s u l t can cause c a n c e r . G i v e n t h a t g e n o t o x i c agents e x e r t t h e i r a c t i v i t y t h r o u g h i r r e v e r s i b l e r e a c t i o n s w i t h n u c l e o p h i l i c atoms ( a d d u c t s ) , the amount o f such r e a c t i o n p r o d u c t s w i l l be p r o p o r t i o n a l not o n l y t o the i n v i v o c o n c e n t r a t i o n o f the e l e c t r o p h i l e , b u t a l s o t o the time t h i s c o n c e n t r a t i o n i s m a i n t a i n e d . T h e r e f o r e , the amount o f m e t a b o l i t e bound t o c e l l u l a r DNA ( i n v i v o d o s e ) , p r o v i d e s a r e l i a b l e d o s i m e t r i c b a s i s upon w h i c h t o a s s e s s the r i s k c o n n e c t e d w i t h exposure t o a g e n o t o x i c compound (3,7,8). I t s h o u l d be n o t e d t h a t the d e t e c t i o n and q u a n t i t a t i o n o f DNA adducts i s not a s i m p l e t a s k because t h e r e are few a n a l y t i c a l t e c h n i q u e s c u r r e n t l y a v a i l a b l e . The reasons f o r t h i s a r e : (a) the a s s a y o f t e n has to accommodate the unique c h e m i c a l and/or p h y s i c a l p r o p e r t i e s o f the i n d i v i d u a l compound or i t s adduct; (b) the p e r c e n t a g e o f t o t a l c h e m i c a l t h a t becomes a t t a c h e d to the DNA i n the t a r g e t t i s s u e i s q u i t e s m a l l because most o f i t i s u s u a l l y d e t o x i f i e d and e x c r e t e d ; (c) not a l l adducts t h a t form between the g e n o t o x i c agent and DNA are s t a b l e o r are i n v o l v e d i n the development o f subsequent d e l e t e r i o u s events i n the organism; and, (d) the amount o f DNA a v a i l a b l e f o r a n a l y s i s from the t a r g e t t i s s u e o r organ i s o f t e n q u i t e l i m i t e d . R e c e n t l y our l a b o r a t o r y demonstrated an a l t e r n a t i v e method t o r a d i o m e t r i c , immuno, or p o s t d e r i v a t i z a t i o n d e t e c t i o n o f the p o l y c y c l i c a r o m a t i c h y d r o c a r b o n , b e n z o [ a j p y r e n e (BaP) b i n d i n g t o DNA ( 9 ) . E s s e n t i a l l y , the t e c h n i q u e c o n s i s t s o f the a c i d - i n d u c e d removal o f the d i o l e p o x i d e adduct o f BaP from the macromolecule as the s t r o n g l y f l u o r e s c e n t f r e e t e t r o l s w h i c h are t h e n s e p a r a t e d and q u a n t i t a t e d by f l u o r e s c e n c e / H P L C a n a l y s i s . The r e s u l t i n g c h r o m a t o g r a p h i c p r o f i l e can be used t o e s t a b l i s h the s t e r e o c h e m i c a l o r i g i n o f the d i o l e p o x i d e i n v o l v e d i n adduct f o r m a t i o n ( 1 0 ) . Benzofalpyrene-DNA Adduct D e t e c t i o n . Our i n i t i a l r e s e a r c h w i t h the f l u o r e s c e n c e / H P L C t e c h n i q u e has demonstrated t h a t we can d e t e c t , i d e n t i f y , and q u a n t i t a t e , subsequent t o s e v e r a l r o u t e s o f exposure, the b i n d i n g o f BaP w i t h c e l l u l a r DNA o f mice (10-12) and f i s h (13.) . The d e t e c t i o n and q u a n t i t a t i o n o f adduct f o r m a t i o n i n f i s h exposed t o BaP demonstrate t h a t a q u a t i c s p e c i e s are s i m i l a r to o t h e r organisms i n t h a t they p o s s e s s c e l l u l a r enzymes c a p a b l e o f m e t a b o l i z i n g BaP to the u l t i m a t e c a r c i n o g e n i c form o f the c h e m i c a l , the d i o l e p o x i d e . A comparison o f BaP-adducts formed w i t h the DNAs o f mice and f i s h demonstrate s i m i l a r i t i e s (Table I I ) , and s u g g e s t the f e a s i b i l i t y o f e x t r a p o l a t i o n o f exposure d a t a , b a s e d on the d e t e c t i o n and q u a n t i t a t i o n o f DNA adduct l e v e l s , between s p e c i e s . F i e l d S t u d i e s w i t h B e l u g a Whale. Large q u a n t i t i e s o f PAHs, i n c l u d i n g the s t r o n g l y c a r c i n o g e n i c BaP, have been r e p o r t e d i n the main h a b i t a t o f the S t . Lawrence b e l u g a whale p o p u l a t i o n . These compounds are the p r o b a b l e cause o f the i n c r e a s e d f r e q u e n c y o f


7. SHUGARTETAL.

89


T a b l e I I . Comparison o f DNA Adduct F o r m a t i o n i n t h e Mouse and B l u e g i l l S u n f i s h a f t e r an A c u t e Exposure t o Benzo|[ajpyrene


Species

Macromolecule

DNA Adduct F o r m a t i o n * 1-1 II-2 %I-1

Mice

S k i n DNA**

588

220

73

Fish

L i v e r DNA***

928

127

88

* DNA adduct f o r m a t i o n d e t e r m i n e d 72 hours subsequent t o BaP exposure and e x p r e s s e d as nanograms o f t e t r o l 1-1 o r I I - 2 p e r gram o f DNA. Data t a k e n from (10-13). ** T o p i c a l a p p l i c a t i o n o f BaP a t 3 micrograms p e r gram body w e i g h t . *** I n t r a p e r i t o n e a l i n j e c t i o n o f BaP a t 5 micrograms p e r gram body w e i g h t . F i s h m a i n t a i n e d a t 20 C. o

b l a d d e r tumors seen among aluminum workers from t h e same a r e a ( 1 4 ) , and s i n c e t h e compounds contaminate t h e b e l u g a whale f o o d c h a i n , t h e y a r e c o n s i d e r e d t o be t h e e t i o l o g i c a l f a c t o r f o r t h e h i g h f r e q u e n c y o f tumors seen i n t h i s p o p u l a t i o n ( 1 5 ) . D e t e c t a b l e l e v e l s o f BaP-DNA adducts were found i n t h e b r a i n s o f b e l u g a whales from t h e S t . Lawrence e s t u a r y , w h i l e t h e o c c u r r e n c e o f BaP-DNA adducts o f t h e b r a i n and l i v e r o f b e l u g a s t a k e n from t h e Mackenzie e s t u a r y was n o t o b s e r v e d ( T a b l e I I I ) . Table I I I .

D e t e c t i o n o f Benzo[a]pyrene Adducts i n DNA o f B e l u g a whales ( D e l p h i n a p t e r u s l e u c a s )

Sample

Tissue

BaP Adduct F o r m a t i o n binding* level**

S t . Lawrence E s t u a r y #1 #2 #3

Brain Brain Brain

206 94 69

Mackenzie E s t u a r y #1 - #4

Brain

none d e t e c t e d

#1 - #4

Liver

none d e t e c t e d

* per ** 10 7

2. 15 0. 98 0. 73

BaP adducts t o DNA e x p r e s s e d as nanograms o f t e t r o l 1-1 gram o f DNA. Data t a k e n from ( 1 5 ) . L e v e l o f adduct f o r m a t i o n e x p r e s s e d as BaP adducts p e r DNA n u c l e o t i d e s .



90


The d a t a o b t a i n e d a r e i m p o r t a n t f o r s e v e r a l r e a s o n s . F i r s t , they p r o v i d e e v i d e n c e t h a t t h e whales from t h e S t . Lawrence e s t u a r y had been exposed t o BaP, and had m e t a b o l i z e d i t t o t h e d i o l e p o x i d e , w h i c h s u b s e q u e n t l y became c o v a l e n t l y bound t o t h e DNA o f t h e b r a i n t i s s u e . Over t h e p a s t s e v e r a l y e a r s a s t r o n g and c o n v i n c i n g argument has d e v e l o p e d f o r a c a u s a l r e l a t i o n s h i p between t h e c a r c i n o g e n i c potency o f BaP and t h e amount bound t o an organism's DNA as a r e s u l t o f c e l l u l a r metabolism ( 6 , 1 6 ) . T h i s i s r e i n f o r c e d by t h e premise t h a t t h e i n t e g r i t y o f DNA i s e s s e n t i a l f o r s u r v i v a l . A l t e r a t i o n s , i f l e f t u n c o r r e c t e d , c o u l d t r i g g e r a sequence o f e v e n t s t h a t c u l m i n a t e s i n t h e appearance o f an o v e r t m a l i g n a n c y . Second, t h e l e v e l o f adducts d e t e c t e d approaches t h a t found i n a n i m a l s , b o t h t e r r e s t r i a l and a q u a t i c , exposed under c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s t o a c a r c i n o g e n i c dose o f BaP (12,13,17,18). T h i r d , c e l l s i n b r a i n t i s s u e a r e known t o have slow t u r n o v e r r a t e s and l a c k o r have v e r y low c a p a c i t y f o r e x c i s i o n r e p a i r o f DNA damage. T h e r e f o r e , s i g n i f i c a n t a c c u m u l a t i o n o f DNA adducts c o u l d o c c u r i n t h i s t i s s u e from l o n g - t e r m c h r o n i c exposure t o e n v i r o n m e n t a l l e v e l s o f BaP t o o low t o i n d u c e n e o p l a s i a ( 1 9 ) . I t s h o u l d be n o t e d t h a t t h e h e a l t h o f an a n i m a l may i n f l u e n c e t h e f o r m a t i o n o f a d d u c t s . The whales from t h e Mackenzie e s t u a r y were h u n t e d a n i m a l s and n o t known t o be d i s e a s e d a t t h e time o f t h e i r c a p t u r e , w h i l e those from t h e S t . Lawrence were beached a n i m a l s . Non-adductive DNA Damage. C e r t a i n g e n o t o x i c compounds o r agents such as m e t a l s , r a d i o n u c l i d e s , some c h e m i c a l s , e t c . , do n o t c o v a l e n t l y b i n d t o DNA, b u t n e v e r t h e l e s s i n d u c e damage. I f t h i s damage i s e x p r e s s e d as s t r a n d breakage ( o r t h e p o t e n t i a l f o r s t r a n d b r e a k a g e ) , i t c a n be d e t e c t e d by measuring t h e r a t e o f a l k a l i n e i n d u c e d s t r a n d s e p a r a t i o n (20) . A n a l y s i s by a m o d i f i e d a l k a l i n e u n w i n d i n g a s s a y (21) demonstrated t h a t c h e m i c a l l y i n d u c e d DNA s t r a n d breakage c o u l d be d e t e c t e d i n b l u e g i l l s u n f i s h and f a t h e a d minnows exposed t o c h r o n i c , low l e v e l s o f BaP. T h i s t e c h n i q u e has been u s e d t o determine t h e r e l a t i v e DNA damage, as measured by s t r a n d breakage, i n b l u e g i l l s u n f i s h t a k e n from s e v e r a l s i t e s i n White Oak Creek, w h i c h i s a s m a l l stream t h a t f l o w s t h r o u g h t h e Department o f Energy r e s e r v a t i o n a t t h e Oak Ridge N a t i o n a l L a b o r a t o r y and t e r m i n a t e s i n White Oak Lake. B i o m o n i t o r i n g a c t i v i t i e s a r e b e i n g c o n d u c t e d i n t h i s system ( 2 7 ) . The d a t a i n T a b l e IV show t h a t t h e i n t e g r i t y o f t h e DNA o f t h e f i s h i n t h e White Oak Creek System, e x c e p t f o r those a t t h e 3.0-3.4 km s i t e , i s s i m i l a r t o t h a t o f f i s h t a k e n from a c o n t r o l s i t e (Brushy F o r k Creek, which i s l o c a t e d some d i s t a n c e from t h e ORNL r e s e r v a t i o n ) f o r b o t h t h e S p r i n g and F a l l c o l l e c t i o n t i m e s . A d d i t o n a l s t u d i e s w i t h f i s h c o l l e c t e d a t t h e 3.0-3.4 km s i t e a r e i n p r o g r e s s . These d a t a , a l t h o u g h p r e l i m i n a r y , i n d i c a t e t h e p o t e n t i a l f o r measuring DNA damage as a g e n e r a l b i o l o g i c a l marker o f exposure o f i n d i g e n o u s a n i m a l s t o contaminants t h a t o c c u r i n t h e i r environment. Hemoglobin M o d i f i c a t i o n . Because i t meets a number o f e s s e n t i a l r e q u i r e m e n t s , hemoglobin has been proposed as an a l t e r n a t i v e c e l l u l a r macromolecule t o DNA f o r e s t i m a t i n g t h e i n v i v o dose o f


7. SHUGARTETAL.

T a b l e IV.

R e l a t i v e DNA S t r a n d Breaks i n B l u e g i l l from the White Oak Creek Watershed

Site

n* Spring


91


W.O.C.(3.0-3.4 km) W.0.C.(2.6 km) White Oak Lake Brushy F o r k Creek

1987

4.77 0.68 0.75 0.91

F a l l 1987

2..01 0..03 1..13 1..15

* n - ( I n F / l n F ) - 1 - number o f b r e a k s p e r a l k a l i n e u n w i n d i n g u n i t o f DNA (see r e f s 20.21). F r e p r e s e n t s f r a c t i o n o f DNA i n double s t r a n d e d form from f i s h a t sample s i t e s l i s t e d . F repres e n t s f r a c t i o n o f DNA i n double s t r a n d e d form from f i s h a t M e l t o n Branch, a s m a l l stream t h a t e n t e r s the White Oak Creek (W.O.C.) a t 2.5 km. A minimum o f f i v e f i s h were a n a l y z e d a t each s i t e . s

c

s

c

c h e m i c a l s subsequent t o exposure (22.-25.). F i r s t , i t has r e a c t i v e n u c l e o p h i l i c s i t e s and the r e a c t i o n p r o d u c t s w i t h e l e c t r o p h i l i c agents a r e s t a b l e . Over f i f t y compounds t o date have been shown t o y i e l d c o v a l e n t r e a c t i o n p r o d u c t s w i t h hemoglobin i n a n i m a l e x p e r i m e n t s . These compounds i n c l u d e r e p r e s e n t a t i v e s from most o f the i m p o r t a n t c l a s s e s o f g e n o t o x i c c h e m i c a l s c u r r e n t l y known. No t e s t e d mutagenic o r c a n c e r - i n i t i a t i n g compound has f a i l e d t o produce c o v a l e n t r e a c t i o n p r o d u c t s w i t h hemoglobin. Secondly, hemoglobin has a w e l l e s t a b l i s h e d l i f e - s p a n , i s r e a d i l y a v a i l a b l e i n humans and a n i m a l s , and i t s c o n c e n t r a t i o n i n an i n d i v i d u a l i s not subject to large v a r i a t i o n . T h i r d l y , m o d i f i c a t i o n of hemoglobin has been shown t o g i v e an i n d i r e c t measure o f the dose o f r e a c t i o n p r o d u c t t h a t b i n d s t o the DNA i n the t a r g e t c e l l s . B i o m o n i t o r i n g o f hemoglobin-bound m e t a b o l i t e s r e p r e s e n t s a n o v e l approach t o the e s t i m a t i o n o f exposure t o p o t e n t i a l l y h a r m f u l c h e m i c a l s ( 2 6 ) . A l t h o u g h these adducts have no p u t a t i v e m e c h a n i s t i c r o l e i n c a r c i n o g e n e s i s , t h e y do r e l a t e q u a n t i t a t i v e l y t o exposure and t o a c t i v a t i o n s i n c e t h e y may approximate the s y s t e m i c dose ( i n v i v o dose) o f a c h e m i c a l . They can be a measure o f the c h e m i c a l ' s g e n o t o x i c potency as w e l l . Furthermore, s i n c e the adducts w h i c h form w i t h hemoglobin a r e s t a b l e and have l i f e spans e q u a l t o t h a t o f the c i r c u l a t i n g e r y t h r o c y t e ( 2 3 ) , q u a n t i t a t i o n o f these adducts can be used t o i n t e g r a t e the dose o b t a i n e d from c h r o n i c l o w - l e v e l types o f exposure w h i c h most l i k e l y o c c u r i n c h e m i c a l l y - p o l l u t e d environments. F i e l d S t u d i e s w i t h T e r r e s t r i a l A n i m a l s . A f l o o d p l a i n on E a s t F o r k P o p l a r Creek (EFPC) was s e l e c t e d as a s t u d y s i t e . EFPC o r i g i n a t e s a t New Hope Pond, a p o i n t source o f i n d u s t r i a l p o l l u t i o n , and the s t u d y s i t e i s about 4 k i l o m e t e r s downstream. The f l o o d p l a i n i s low and the c r e e k p e r i o d i c a l l y o v e r f l o w s , d e p o s i t i n g sediment. There



92


i s abundant v e g e t a t i o n . Measured BaP c o n c e n t r a t i o n s i n the s o i l was 70 nanograms/gram, a l t h o u g h 10 times t h i s amount have been r e p o r t e d . C o n c e n t r a t i o n s i n p l a n t s was l e s s than l / 1 0 t h o f t h a t i n the s o i l . S m a l l animals were t r a p p e d a t t h i s s i t e t o determine the p r a c t i c a l i t y o f u s i n g hemoglobin as a s u i t a b l e b i o l o g i c a l marker o f BaP p o l l u t i o n . P r e l i m i n a r y d a t a (Table V) i n d i c a t e d e t e c t a b l e c o n c e n t r a t i o n s of BaP adducts w i t h hemoglobin were p r e s e n t i n some i n d i v i d u a l s o f s p e c i e s which have i n t i m a t e c o n t a c t w i t h the sediment o r s o i l . The s h o r t t a i l shrew burrows i n t o the ground and e a t s earthworms and i n s e c t s , w h i l e muskrats f e e d on i n v e r t e b r a t e s found i n the c r e e k bed and browse on muddy v e g e t a t i o n a l o n g the c r e e k bank. BaP adducts were n o t d e t e c t a b l e i n h e r b i v o r e s such as the w h i t e - f o o t e d mouse. T a b l e V.

C o n c e n t r a t i o n o f Benzo[a]pyrene Adducts i n the Hemoglobin o f Animals on the EFPC F l o o d p l a i n

Species

Number Analyzed

W h i t e - f o o t e d mouse (Peromvscus leucopus) House mouse (Mus muscuius) S h o r t t a i l shrew ( B l a r i n a brevicauda) B l a c k r a t snake (Elaphe o b s o l e t a o b s o l e t a ) Snapping t u r t l e (Chelvdra serpentina) Norway r a t (Rattus norvepicus) Muskrat (Ondatra z i b e t h i c a )

BaP-hemoglobin Adducts*

15

0,.00

1

0..00

2

0,.35

1

0..00

1

0,.16

1

0,.15

6

0,.23

* BaP adducts t o hemoglobin e x p r e s s e d as nanograms o f t e t r o l 1-1 p e r gram o f hemoglobin. Data t a k e n from ( 2 7 ) . BaP-hemoglobin Adducts i n Humans. Human d a i l y exposure t o BaP i n c r e a s e s d r a m a t i c a l l y i n smokers, as one c i g a r e t t e c o n t a i n s between 10 and 50 nanograms o f BaP. S i n c e the l i f e - s p a n o f a human red b l o o d c e l l i s s e v e r a l months, c o n t i n u a l exposure t o BaP a t one or more packs o f c i g a r e t t e s p e r day s h o u l d r e s u l t i n a measurable s t e a d y s t a t e c o n c e n t r a t i o n o f BaP-hemoglobin a d d u c t s . A p r e l i m i n a r y s t u d y was conducted i n a l i m i t e d p o p u l a t i o n o f humans to determine whether BaP-hemoglobin adducts c o u l d be d e t e c t e d . The d a t a i n T a b l e VI (methodology a c c o r d i n g t o (25,26)) show t h a t adducts were p r e s e n t i n f i v e out o f s i x i n d i v i d u a l s who smoked w h i l e s i m i l a r a n a l y s e s o f non-smokers gave lower l e v e l s r e l a t i v e t o smokers. These d a t a demonstrate the f e a s i b i l i t y o f the t e c h n i q u e ,


7. SHUGARTETAL.


93

however, i n t r i n s i c f a c t o r s such as age, sex, h e a l t h , and n u t r i t i o n a l s t a t u s , as w e l l as e x t r i n s i c f a c t o r s such as t h e p r e s e n c e o f o t h e r c h e m i c a l s , may i n f l u e n c e t h e p h a r m a c o k i n e t i c s o f BaP metabolism, and t h e i n c i d e n c e o f adducts w i t h hemoglobin.


Table VI.

B e n z o [ a j p y r e n e Adducts i n Hemoglobin o f Smokers and Non-smokers

Sample No. Sex Age Race

1 2 3 4 5 6 7 8 9 10 11 12 13 14

m m f f m m f m m m f m f m

23 44 54 56 36 40 28 42 49 27 56 49 50 57

C B C C 0 C C C 0 B C C C C

Smoking Habit*

none none 20 none none f o r 8 y r s none f o r 15 y r s none 20 40 none none f o r 1 y r 20 40 40+

BaP-hemoglobin Adducts**

64 24 nd 69 73 nd nd 24 159 129 1402*** 468 272 229

C i g a r e t t e s p e r day. ** BaP adducts t o hemoglobin e x p r e s s e d as picograms o f t e t r o l 1-1 p e r gram o f hemoglobin; nd: none d e t e c t e d . *** Numerous f l u o r e s c e n t peaks p r e s e n t i n chromatogram i n a d d i t i o n t o t e t r o l 1-1 peak. X e n o b i o t i c M e t a b o l i s m . X e n o b i o t i c s i n t h e environment, i f t a k e n up by an organisms, may induce microsomal enzymes (mixed f u n c t i o n o x i d a s e s , MFO's) t h a t c a n d e t o x i f y many o f t h e s e compounds (28.). The MFO's c a t a l y z e a v a r i e t y o f r e a c t i o n s , i n c l u d i n g a r o m a t i c and a l i p h a t i c N - h y d r o x y l a t i o n , N - d e a l k y l a t i o n , and O - d e a l k y l a t i o n . P a r a d o x i c a l l y , t h e MFO's may a c t i v a t e c e r t a i n c h e m i c a l s t o t h e i r c a r c i n o g e n i c m e t a b o l i t e ( s ) ( 2 9 ) . The b i o c h e m i c a l systems i n f i s h r e s p o n s i b l e f o r t h e s e types o f r e a c t i o n s a r e s i m i l a r t o t h o s e found i n mammalian s p e c i e s (30,31). The use o f enzyme a s s a y s i n b i o l o g i c a l m o n i t o r i n g programs was r e v i e w e d by Payne (32) f o r d i f f e r e n t p h y l a o f a q u a t i c a n i m a l s and was found t o be u s e f u l i n d e t e c t i n g known p o i n t s o u r c e s o f p o l l u t i o n . F i e l d S t u d i e s i n A q u a t i c Organisms. A c t i v i t i e s o f s e v e r a l MFO parameters have been measured i n f i s h c o l l e c t e d from s t a t i o n s a l o n g E a s t F o r k P o p l a r Creek ( 3 3 ) . A m u l t i v a r i a t e d i s c r i m i n a n t a n a l y s i s was used t o examine t h e p a t t e r n o f s e v e r a l measures o f t h e MFO system ( e t h o x y r e s o r u f i n d e e t h y l a s e , NAD r e d u c t a s e , NADP r e d u c t a s e ,



94


cytochrome P-450, and cytochrome D5 l e v e l s ) i n f i s h from the d i f f e r e n t s t a t i o n s . The s t a t i s t i c a l a n a l y s i s ( F i g u r e 1) c o n f i r m t h a t female b l u e g i l l s u n f i s h a t s t a t i o n n o . l (EFPC a t New Hope Pond), a s o u r c e o f i n d u s t r i a l p o l l u t i o n , e x h i b i t e d a s i g n i f i c a n t l y d i f f e r e n t p a t t e r n o f MFO responses t h a n f i s h c o l l e c t e d 20 k i l o m e t e r s down stream ( s t a t i o n no. 5) o r from a p r i s t i n e stream (control). In addition, histopathological analysis of various organs from f i s h a l o n g EFPC showed h i g h e r l e v e l s o f l e s i o n s i n the f i s h closest to s t a t i o n n o . l . These r e s u l t s ( F i g u r e 1) suggest t h a t the MFO enzyme system can be u s e f u l as a b i o l o g i c a l marker i n a m o n i t o r i n g program i f the response o f the system t o e n v i r o n m e n t a l and p h y s i o l o g i c a l i n f l u e n c e s such as temperature, season, f o o d , hormonal ( s e x ) , e t c . , are taken i n t o c o n s i d e r a t i o n . Summary and C o n c l u s i o n s E f f e c t i v e e n v i r o n m e n t a l management r e q u i r e s knowledge o f the t r a n s p o r t and f a t e o f contaminants i n n a t u r a l systems ( 3 4 ) . S m a l l mammals can s e r v e as i n d i c a t o r s o f the presence and b i o a v a i l a b i l i t y o f contaminants and s e l e c t e d m o n i t o r i n g s p e c i e s can be used t o measure the a c c u m u l a t i o n o f c o n t a m i n a n t s . Furthermore, knowledge o f f o o d h a b i t s and h a b i t a t may i n d i c a t e pathways o f c o n t a m i n a n t s t o organisms M o n i t o r i n g b i o l o g i c a l e n d p o i n t s i n w i l d a n i m a l s can be used as an a l t e r n a t i v e t o e x t e n s i v e s a m p l i n g and c h e m i c a l a n a l y s e s , p a r t i c u l a r l y where a l a r g e a r e a i s i n v o l v e d and the o r i g r n o r e x t e n t o f c o n t a m i n a t i o n i s unknown o r p o o r l y d e f i n e d . E v i d e n c e o f exposure o f b i o t a as d e t e r m i n e d by b i o l o g i c a l m o n i t o r i n g c o u l d h e l p t o e s t a b l i s h the need f o r c l e a n u p . Our l a b o r a t o r y has been i n t e r e s t e d i n d e v i s i n g methods f o r d e t e c t i n g the exposure o f l i v i n g organisms t o hazardous c h e m i c a l s and p h y s i c a l agents i n the environment. I n i t i a l studies with c h e m i c a l d o s i m e t r y a n a l y s i s began w i t h the development o f a f l u o r e s c e n c e / H P L C a s s a y f o r the d e t e c t i o n and q u a n t i t a t i o n o f BaPDNA adducts w i t h a s e n s i t i v i t y s u f f i c i e n t f o r d e t e c t i n g e n v i r o n m e n t a l exposures. Subsequent r e s e a r c h demonstrated the a p p l i c a b i l i t y o f t h i s t e c h n i q u e t o a n i m a l models, b o t h a q u a t i c and t e r r e s t r i a l , under v a r i o u s e x p e r i m e n t a l c o n d i t i o n s . Furthermore, hemoglobin m o d i f i c a t i o n by c h e m i c a l s was shown t o be e q u i v a l e n t t o DNA b i n d i n g as a d o s i m e t e r . S t u d i e s i n a q u a t i c organisms, b o t h i n the l a b o r a t o r y and i n the f i e l d , have demonstrated the f e a s i b i l i t y o f measuring MFO enzyme a c t i v i t i e s a s s o c i a t e d w i t h the metabolism o f x e n o b i o t i c s , such as BaP, i n c o n j u n c t i o n w i t h n o n - a d d u c t i v e DNA damage as p o t e n t i a l l y u s e f u l b i o m a r k e r s f o r exposure and b i o a v a i l a b i l i t y o f e n v i r o n m e n t a l pollutants. Other b i o l o g i c a l markers a r e c u r r e n t l y b e i n g i n v e s t i g a t e d f o r t h e i r c a p a c i t y t o d e t e c t and q u a n t i t a t e exposure t o c o n t a m i n a n t s . These i n c l u d e : (a) parameters o f the immune response ( i n t e r f e r o n i n d u c t i o n , a n t i b o d y - f o r m i n g c e l l s , and lymphocyte response t o m i t o g e n s ) ; (b) i n d u c t i o n o f m e t a l - b i n d i n g p r o t e i n s ; and, (c) minor n u c l e o s i d e c o n t e n t o f DNA.



SHUGART ET AL.


4 r-

UJ

3

_

FEMALE BLUEGILLS (WINTER)

J

-5

-4

-3

I

I

I

-2 -1 0 1 2 FIRST CANONICAL VARIABLE

L

3

4

5

F i g u r e 1. S e g r e g a t i o n o f i n t e g r a t e d v a r i a b l e groups by d i s c r i m i n a n t a n a l y s i s u s i n g s e v e r a l measures o f t h e MFO system. C i r c l e s r e p r e s e n t 95% c o n f i d e n c e r a d i i o f c o n t r o l and r e s p e c t i v e t e s t s t a t i o n s . S t a t i o n no. 1 (New Hope Pond) i s t h e headwaters o f E a s t Fork P o p l a r Creek; i n c r e a s i n g s t a t i o n numbers a r e f u r t h e r down stream. Data t a k e n from ( 3 3 ) .


96


Acknowledgments


Portions of this work were sponsored by the Department of Environmental Management in the Health, Safety, Environment and Accountability Division of the Oak Ridge Y-12 Plant; and the ORNL Director's R&D Program. The Oak Ridge Y-12 Plant and the Oak Ridge National Laboratory are operated by Martin Marietta Energy Systems, Inc., under contract DE-AC05-84OR21400. Environmental Sciences Division, Oak Ridge National Laboratory Publication No. 3088. Literatute Cited 1. Beardsley, A., Vogg, J., Beckett, P.H.T., and Sanso, B.F. Environ. Pollut. 1978, 16, 65-71. 2. Maugh, T.H. Science 1984, 219, 1032-1033. 3. Perera, F.P., Poirer, M.C., Yuspa, S.H., Nakayama, J., Jaretski, A., Curnern, M.M., Knowles, D.M., and Weinstein, I.B. Carcinogenesis 1982, 3, 1405-1410. 4. Montesano, R., Rajewksy, M.F., Pegg, A.F., and Miller, E. Cancer Res. 1982, 42, 5236-5239. 5. Gelboin, H., and T'so, P.O.P. Polycyclic Hydrocarbons and Cancer: Molecular Biology and Environment: Academic: New York, 1979. 6. Harvey, R.G. Am. Scientist 1982, 70, 386-393. 7. Wogan, G.N., and Gorelick, N.J. Environ. Health Perspec. 1985, 62, 5-18. 8. Shamsuddin, A.K.M., Sinopoli, N.J., Hemminki, K., Boesch, R.R., and Harris, C.C. Cancer Res. 1985, 45, 66-68. 9. Rahn, R., Chang, S., Holland, J.M., and Shugart, L.R. Biochem. Biophys. Res. Commun. 1982, 109, 262-269. 10. Shugart, L.R., Holland, J.M., and Rahn, R. Carcinogenesis 1983, 4, 195-199. 11. Shugart, L.R., J. Toxicol. Environ. Health 1985, 15, 255-263. 12. Shugart, L.R., and Kao, J. Environ. Health Perspec. 1985, 62, 223-226. 13. Shugart, L.R., McCarthy, J.F., Jimenez, B.D., and Daniel, J. Aquatic Toxicol. 1987, 9, 319-325. 14. Theriault, G., and Tremblay, C. The Lancet 1984, 1, 947-950. 15. Martineau, D., Legace, A., Beland, P., Higgins, R., Armstrong, D., and Shugart, L.R. Proc. 7th Biennial Conf. on Biology of Marine Mammals 1987, p 45. 16. Phillip, D.H. Nature 1983, 303, 468-472. 17. Varanasi, U., Stein, J.E., and Hom, T. Biochem.Biophys.Res. Commun. 1981, 103, 780-787. 18. Stowers, S.J., and Anderson, M.W. Chem.-Biol. Interact. 1984, 51, 151-166. 19. Stowers, S.J., and Anderson, M.W. Environ. Health Perspec. 1985, 62, 31-39. 20. Rydberg, B. Rad. Res. 1975, 61, 274-287. 21. Shugart, L.R. Mar. Environ. 1988, 24, 321-325. 22. Osterman-Golkar, S., Ehrenberg, L., Segebrack, D., and Halstrom, I. Mut. Res. 1976, 34, 1-20. 23. Calleman, C.J. Ph.D. Thesis Department of Radiobiology, University of Stockholm, Sweden, 1984.


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