In Vivo and Biochemical Determination of Toxic Cadmium in Rats

Chapter 16

In Vivo and Biochemical Determination of Toxic Cadmium in Rats 1

Chien Chung and Wen-Kang Chen

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Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0445.ch016

1

Institute of Nuclear Science, National Tsing Hua University, Hsinchu 30043, Taiwan Department of Biochemistry, Chung-Shan Medical and Dental College, Taichung 40203, Taiwan

2

The f e a s i b i l i t y of i n vivo prompt gamma-ray a c t i v a t i o n of toxic Cd concentrations i n poisoned rats has been demonstrated by p a r t i a l body i r r a d i a t i o n with minimal radiation doses using a small mobile reactor neutron beam. Rats chronically exposed to toxic Cd are diagnosed i n vivo and followed by biochemical t e s t s . The results of biochmical tests did not correlate to the Cd concentration except when the rats were f a t a l l y poisoned. Features of the i n vivo determination of whole body Cd concentration i n rats are discussed.

Cadmium is one of the toxic heavy metals which i s used i n various industries; it i s absorbed occupationally by workers v i a inhalation of polluted a i r or ingestion of contaminated food and water. The initial Cd uptake i s transported from the lungs or g a s t r o i n t e s t i n a l t r a c t by the blood to the l i v e r and kidneys(1). Prolonged exposure to Cd eventually leads to kidney problems, anemia, hypertension, and l i v e r damage(2,3). In general, b i o l o g i c a l f l u i d s such as urine and blood are analyzed to deduce the toxic Cd concentration(4). Since Cd exists only i n trace amount i n the urine and blood, i n v i t r o analysis may not show the quantity of Cd i n the body or i n the organ d i r e c t l y ( 5 ) . Thus, Cd l e v e l i n blood and urine cannot be used for predicting possible nephropathy. For example, cadmium i n the blood of exposed workers did not correlate with any biochemical parameter that would indicate potential renal dysfunction(6). Recently, i n vivo prompt

0097-6156/91/0445-0215$06.00/0 © 1991 American Chemical Society

In Biological Trace Element Research; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

216

BIOLOGICAL TRACE ELEMENT RESEARCH


gamma neutron a c t i v a t i o n analysis(IVPGAA) became a v a i l a b l e t o d i r e c t l m e a s u r e t h e Cd c o n c e n t r a t i o n i n human organs, t h e r e b y making e a r l y t h e r a p y p o s s i b l e ( 7 ) . The concept o f IVPGAA was f i r s t i n t r o d u c e d by J . Anderson(8), f o l l o w e d by o t h e r i n v e s t i g a t o r s ( 9 , 1 0 ) . I n Taiwan, IVPGAA study on l i v i n g b e i n g s by t h e use o f n u c l e a r r e a c t o r was begun i n 1983 a t t h e N a t i o n a l T s i n g Hua u n i v e r s i t y ( 1 1 - 1 5 ) . An e x t e n s i v e i n v e s t i g a t i o n i n e v a l u a t i n g r a d i a t i o n s a f e t y and phantom-simulated t e s t s has been completed u s i n g r e a c t o r n e u t r o n beams(16). In t h i s work, r a t s c h r o n i c a l l y p o i s o n e d by Cd as w e l l as a c o n t r o l l e d group a r e diagnosed w i t h both t h e IVPGAA t e c h n i g u e and b i o c h e m i c a l t e s t s . The r e s u l t s o b t a i n e d may s e r v e as a b a s i s f o r f u t u r e c l i n i c a l a p p l i c a t i o n s i n human b e i n g s poisoned by cadmium. EXPERIMENTAL DETERMINATION OF CADMIUM IN RATS To perform t h e i n v i v o m e d i c a l d i a g n o s i s and b i o c h e m i c a l b l o o d t e s t , 19 Long-Evans r a t s were used. They were kept i n a c o n t r o l l e d atmosphere where t h e temperature was 2 2 ± 1 ° C w i t h 50 + 5% h u m i d i t y and t h e l i g h t / d a r k c y c l e was 12/12 hours a day. They were f e d w i t h water u n t i l f u l l y grown t o 140 + 20 g. The Long-Evans r a t s were d i v i d e d i n t o c h r o n i c - p o i s o n e d and c o n t r o l groups. The c h r o n i c Cd-poisoned group was f e d s t a n d a r d Cd s o l u t i o n ( 2 a g/ml) once a day u n t i l t h e i n j e c t i o n dose reached 200-^875 mg. I n t h e c o n t r o l group, f i v e r a t s were f e d normal f o o d f o r two weeks. The c h r o n i c a l l y Cd-poisoned and normal r a t s were examined u s i n g t h e IVPGAA t e c h n i q u e a s c r i b e d i n o u r p r e v i o u s i n v e s t i g a t i o n ( 1 7 ) a t t h e m o d i f i e d c r i t i c a l assembly o f t h e T s i n g Hua M o b i l e E d u c a t i o n a l Reactor(THMER) facility(18). Each l i v i n g r a t d e s t i n e d f o r whole-body i r r a d i a t i o n was l o c k e d i n an a c r y l i c box l i n e d w i t h t i s s u e paper. When t h e neutron beam from THMER was d i r e c t e d a t t h e l i v i n g r a t , a 559 keV prompt gamma-ray was produced, measured u s i n g a gamma-ray s p e c t r a l system. The prompt gamma-ray spectrum was s t o r e d , a n a l y z e d , and i t s a b s o l u t e count r a t e was e v a l u t e d . The whole body Cd c o n c e n t r a t i o n i n t h e r a t was deduced u s i n g a c o n v e r s i o n f a c t o r o f 5.285 mg/g/cps o b t a i n e d from t h e c a l i b r a t i o n u s i n g s t a n d a r d CdCl solution injected i n the rat(17). 2

Cadmium p o i s o n i n g i s u s u a l l y diagnosed by a combination of body f l u i d a n a l y s i s f o r Cd and e v a l u a t i o n o f o t h e r b i o c h e m i c a l parameters. T h e r e f o r e , we a n a l y z e d t h e r a t serum t o o b t a i n d a t a i n c r e a t i n i n e , u r i c a c i d , aggregated


15.

ZHE-MING ET AL.

AS, BI, CU, PB, NI, and SE in Biological Samples

p r o t e i n , albumin, b l o o d urea n i t r o g e n ( B U N ) , a l k a l i n e phosphatase(AP), g l u t a m i c p y r u v i c t r a n s a m i n a s e ( A L T ) , and g l u t a m i c o x a l o a c e t i c transaminanse(AST) w i t h a view t o c o r r e l a t e t h e i r v a r i a t i o n w i t h t h e l e v e l o f Cd p o i s o n i n g . RESULT AND

DISCUSSION


In T a b l e I , t h e b a c k g r o u d - s u b t r a c t e d n e t count r a t e o f t h e 559 keV prompt gamma-ray from t h e Cd(n,y )Cd r e a c t i o n i n the Cd-poisoned and normal r a t s as w e l l as t h e deduced

Table I .

Rat

In v i v o D e t e r m i n a t i o n o f Whole Body Cadmium Concentration i n Rats.

Number

a

Count Rate (cps) b

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

1.116(3)° 1.084(3) 1.002(3) 0.948(3) 0.977(3) 0.799(3) 0.762(3) 0.791(3) 0.785(3) 0.744(3) 0.652(3) 0.546(2) 0.374(2) 0.336(2)

15. 16. 17. 18. 19.

0.0003(3) 0.0014(10) 0.004(3) 0.008(3) 0.0004(3)

Whole Body Cd C o n c e n t r a t i o n ( u g/g) 5898(16) 5729(16) 5296(16) 5010(16) 5163(16) 4223(16) 4027(16) 4180(16) 4149(16) 3932(16) 3446(16) 2886(11) 1977(11) 1776(11) 1.6(16) 7.4(53) 21(16) 42(16) 2.1(16)

a.

Rat numbers 1-14 were c h r o n i c a l l y exposed t o Cd and numbers 15-19 were c o n t r o l s n o t exposed t o Cd. b. The 559 keV prompt gamma-ray i n IVPGAA. C. Number i n parenthèse r e p r e s e n t s t h e u n c e r t a i n t y i n t h e last digit. t o t a l body burden a r e l i s t e d . The c h r o n i c a l l y p o i s o n e d r a t s (numbers 1-14) have whole body Cd c o n c e n t r a t i o n r a n g i n g from 1800 t o 5900 u g/g* i n d i c a t i n g n e a r l y a f u l l a b s o r p t i o n from t h e f o o d dosed w i t h Cd. On t h e o t h e r hand, normal r a t s (numbers 15-19) c o n t a i n o n l y a t r a c e amount o f Cd w i t h a body burden range o f 2-40 a g/g/ s i m i l a r t o t h e


217


218


OVERALL Cd CONCENTRATION, Mg/g

F i g . l R e l a t i o n s h i p between whole body Cd c o n c e n t r a t i o n i n r a t t o b i o l o g i c a l i n d i c a t o r o f (a) c r e a t i n i n e , i u / 1 , (b) u r i c a c i d , i u / 1 , (c) aggregated p r o t i n , i u / 1 , (d) albumin, i u / 1 , (e) b l o o d urea n i t r o g e n ( B U N ) , mg/1, ( f ) a l k a l i n e phosphatase(AP), i u / 1 , (g) g l u t a m i c p y r u v i c t r a n s a m i n a s e ( A L T ) , i u / 1 and (h) g l u t m a i c o x a l o a c e t i c t r a n s a m i n a s e ( A S T ) , i u / 1 . Shaded a r e a i n d i c a t e s t h e normal range i n serum.


16.

CHUNG & CHEN

Toxic Cadmium in Rats

219


whole body Cd c o n c e n t r a t i o n i n t h e R e f e r e n c e Man(19). R e s u l t s o f b i o c h e m i c a l t e s t w i t h t h e i r normal r e a d i n g s f o r h e a l t h y r a t s as w e l l as t h e i r c o r r e l a t i o n t o t h e Cd body burden a r e shown i n F i g u r e 1. The b i o c h e m i c a l d a t a f o r r a t s i n c o n t r o l group were a l l w i t h i n t h e normal range o f reported values elsewhere(1). The c r e t i n i n e , u r i c a c i d , a g g r e g a t e d p r o t e i n , and albumin v a l u e s i n f a t a l l y p o i s o n e d r a t s were a l s o w i t h i n t h e normal range, i m p l y i n g such t e s t s were n o t c o r r e l a t e d t o t h e l a r g e i n t a k e o f Cd. On t h e o t h e r hand, i n c r e a s i n g i n t a k e o f Cd t o a f a t a l l e v e l o f 100 a g/g gave abnormnally h i g h v a l u e s o f BUN, AP, ALT, and AST. The c o r r e l a t i o n o f whole body Cd c o n c e n t r a t i o n t o i n v i t r o b i o c h e m i c a l t e s t s i n r a t s i s poor up t o a Cd l e v e l o f 50 /χ g/g. Only a t t h e f a t a l l y poisoned l e v e l o f 100 /x g/g f o r BUN, AP, ALT, and AST i n serum t e s t s gave abnormal values. The r e n a l t a b u l a r damage and organ d y s f u n c t i o n b e g i n t o appear when t h e body burden o f Cd f o r t h e r e n a l c o r t a x i n l i v e r i s >300-400 n g / g ( 2 0 ) . T h e r e f o r e , t h e biochemical information of c r e a t i n i n e , u r i c a c i d , aggregated p r o t e i n , albumin, BUN, AP, ALT, and AST i n serum may n o t y i e l d any c l u e s t o c h r o n i c Cd p o i s o n i n g . CONCLUSION Rats c h r o n i c a l l y exposed t o Cd were diagnosed i n v i v o u s i n g t h e THMER f a c i l i t y . B i o c h e m i c a l t e s t s were performed on serum samples taken from t h e r a t s i n o r d e r t o f i n d p o s s i b l e c o r r e l a t i o n between t h e two r e s u l t s . B i o c h e m i c a l d a t a , however, d i d n o t show any c o r r e l a t i o n t o t h e whole body Cd c o n c e n t r a t i o n ; o n l y when t h e r a t s were f a t a l l y poisoned by a t l e a s t 100 /xg/g o f Cd, b i o c h e m i c a l d a t a f o r BUN, AP, ALT, and AST i n serum y i e l d e d abnormal r e a d i n g s . Thus f o r c h r o n i c a l l y p o i s o n e d r a t s o r p a t i e n t s , b i o c h e m i c a l a n a l y s i s may e a s i l y be misdiagnosed as e v i d e n c e o f p h y s i c a l d i s e a s e o t h e r than Cd poisoning. On t h e c o n t r a r y , t h e i n v i v o prompt gamma a c t i v a t i o n a n a l y s i s c a n diagnose t h e Cd c o n c e n t r a t i o n and, a c c o r d i n g l y , t h e p a t i e n t s can be t r e a t e d p r o p e r l y . ACKNOWLEDGMENTS The a u t h o r s wish t o thank t h e s t a f f o f t h e N u c l e a r S c i e n c e and Technology Development C e n t r e , N a t i o n a l T s i n g Hua U n i v e r s i t y , f o r t h e i r support o f t h i s work. F i n a n c i a l a i d f o r t h i s work was s u p p l i e d by t h e Atomic Energy C o u n c i l o f t h e R e p u b l i c o f China under c o n t r a c t number AEC-0979-025-K2.


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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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RECEIVED July 16, 1990


In Vivo and Biochemical Determination of Toxic Cadmium in Rats

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