Alterations in Rainbow Trout Liver Function and Body Fluids Following

24 Alterations in Rainbow Trout Liver Function and Body Fluids Following Treatment with Carbon Tetrachloride

Downloaded by EAST CAROLINA UNIV on April 7, 2018 | https://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch024

or Monochlorobenzene LAVERN J. WEBER, WILLIAM H. GINGERICH, and KEITH F. PFEIFER Oregon State University Marine Science Center, Marine Drive, Newport, OR 97365 and Department of Fisheries and Wildlife, Oregon State University, Corvallis, OR 97330 It is becoming increasingly evident that drug or chemical induced tissue damage is produced by some foreign compounds through metabolic conversion to a more highly reactive intermediate product. Recent evidence suggests that the mode of hepatic and renal necrosis found following intoxication by certain of the aromatic organic compounds (1, 2); or carbon tetrachloride (CCl )(3) results from activation of the parent compound to a more reactive species. This process of activation is thought to be mediated primarily through a group of mixed function oxidase enzymes which are found in the smooth endoplasmic reticulum. Considerable interest has developed concerning the nature of the mixed function oxidase system in fish and the role that this system may play in the development of toxic responses in these animals. Studies have shown that components of the mixed function oxidase system are present in relatively high concentrations in fish liver (4, 5, 6); and that the enzyme systems in this organ are capable of many of the biotransformation reactions already described for the mammalian liver (7, 8, 9). The presence of this complement of enzymes in the livers of many fishes suggests that this organ too may be particularly sensitive to insult from sublethal concentrations of many waterborne toxicants. For this reason, methods to evaluate liver function in fish may be particularly useful in identifying the sublethal effects of certain classes of toxicants. The use of clinical diagnostic procedures by which to identify and evaluate specific organ dysfunction is used widely in human and mammalian toxicology. Despite the apparent usefulness of such techniques little has been done to develop similar methods for use in studying toxic responses in aquatic organisms. Recently we have evaluated several exogenous and endogenous tests of liver function in rainbow trout following intoxication by the model hepatotoxicant CCl . The results of these studies indicate that elevated plasma activity of the enzyme glutamate-pyruvate transaminase (GPT) is the most sensitive endogenous index of 4

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0-8412-0489-6/79/47-099-401$05.00/0 © 1979 American Chemical Society Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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l i v e r damage i n t r o u t o f t h e s e v e r a l enzyme s y s t e m s t e s t e d ( 1 0 ) . S i m i l a r l y , we have p r e s e n t e d d a t a i n d i c a t i n g t h a t l i v e r f u n c t i o n s t u d i e s b a s e d on p l a s m a c l e a r a n c e o f t h e o r g a n i c a n i o n s u l f o b r o m o p h t h a l e i n (BSP) a l s o a r e u s e f u l i n e v a l u a t i n g l i v e r damage i n r a i n b o w t r o u t ( 1 1 , 1 2 ) . The i n t e n t o f t h e p r e s e n t s t u d y was t o compare and c o n t r a s t two model mammalian h e p a t o t o x i c agents f o r t h e i r p o t e n t i a l t o produce h e p a t o t o x i c i t y i n the r a i n bow t r o u t .


M a t e r i a l s and Methods Rainbow t r o u t (100-250 g) were p u r c h a s e d f r o m R o a r i n g R i v e r f i s h h a t c h e r y , S c i o , Oregon and h e l d u n d e r c o n d i t i o n s p r e v i o u s l y d e s c r i b e d ( 1 1 ) . C h l o r o b e n z e n e (MCB) and c a r b o n t e t r a c h l o r i d e (CCI4) were o b t a i n e d f r o m c o m m e r c i a l s o u r c e s and used w i t h o u t f u r t h e r p u r i f i c a t i o n . C h l o r o b e n z e n e was d i s s o l v e d i n an e q u a l volume o f c o r n o i l and a d m i n i s t e r e d as a s i n g l e i n t r a p e r i t o n e a l ( i . p . ) i n j e c t i o n ( e i t h e r 0.5 o r 1.0 m l / k g ) . C o n t r o l a n i m a l s r e c e i v e d a s i m i l a r volume o f c o r n o i l . Carbon t e t r a c h l o r i d e was g i v e n u n d i l u t e d b y i . p . i n j e c t i o n ( 0 . 2 , 1.0, o r 2.0 ml/kg) and c o n t r o l a n i m a l s r e c e i v e d a s i m i l a r volume o f p h y s i o l o g i c a l saline. A c u t e L e t h a l i t y o f MCB o r C C I 4 . Four groups o f 3 t r o u t were g i v e n MCB u s i n g a dose r a n g e o f f r o m 1.0-3.0 m l / k g . The number o f dead a n i m a l s p e r t r e a t m e n t g r o u p was r e c o r d e d d a i l y f o r 3 days and t h e median l e t h a l dose was c a l c u l a t e d f o r 24, 48 and 72 h b y t h e method o f W e i l ( 1 3 ) . The median l e t h a l dose o f CCI4 was d e t e r m i n e d i n two groups o f 5 f i s h by t h e Up and Down Method o f B r o w n l e e e t a l . (14) u s i n g a dose r a n g e o f f r o m 1.6 t o 5.0 m l / k g . E f f e c t o f I n t o x i c a t i o n by MCB o r CCI4 on P l a s m a C l e a r a n c e o f BSP and P l a s m a A c t i v i t y o f GPT. Twenty f o u r , 48, 72 o r 96 h a f t e r a d m i n i s t r a t i o n o f t h e t o x i c a n t s BSP (5.0 mg/kg) was i n j e c t e d i n t o t h e c a u d a l v e i n o f a n i m a l s i n each t r e a t m e n t g r o u p . A f t e r 45 m i n . f i s h were k i l l e d by a s h a r p b l o w on t h e head and a s i n g l e b l o o d sample was drawn by c a r d i a c p u n c t u r e . Each a n i m a l was w e i g h e d , t h e l i v e r removed and w e i g h e d and s o f t o r g a n s examined f o r g r o s s p a t h o l o g i c a l c h a n g e s . F o r p l a s m a enzyme a c t i v i t y s t u d i e s a s i n g l e b l o o d sample was drawn i n t o a h e p a r i n i z e d t u b e r c u l i n s y r i n g e f r o m t h e c a u d a l v e i n and p l a c e d i n t o a t e s t t u b e r i n s e d w i t h a 2.5% (W/V) s o l u t i o n o f potassium oxalate. E f f e c t o f CCI4 I n t o x i c a t i o n on Water B a l a n c e i n Rainbow Trout. In studies of water balance the u r i n a r y bladder of t r e a t e d and c o n t r o l a n i m a l s was c a t h e t e r i z e d (PE 50 t u b i n g ) and i n d i v i d u a l f i s h were p l a c e d i n p l e x i g l a s s r e s t r a i n i n g chambers f o r 24 h t o a l l o w u r i n e f l o w r a t e s t o s t a b i l i z e . U r i n e was c o l l e c t e d i n t o i n d i v i d u a l t e s t t u b e s a t one h o u r

Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

24.

WEBER E T A L .

Carbon

Tetrachloride

and

Monochlorobenzene

403


intervals. A f t e r 2 4 h f i s h were i n j e c t e d w i t h C C l ^ ( 1 . 0 ml/kg) o r p h y s i o l o g i c a l s a l i n e ( 1 . 0 ml/kg) and u r i n e f l o w r a t e s determined a t h o u r l y i n t e r v a l s f o r 2 4 h. I n a s e p a r a t e group o f f i s h t o t a l plasma p r o t e i n c o n c e n t r a t i o n and plasma o s m o l a l i t y were d e t e r m i n e d f r o m a s i n g l e b l o o d sample drawn from the caudal v e i n . A n a l y t i c a l Methods. The c o n c e n t r a t i o n o f BSP i n t h e p l a s m a was d e t e r m i n e d b y t h e method o f R i c h t e r i c h ( 1 5 ) . The a c t i v i t y o f GPT i n t h e p l a s m a was d e t e r m i n e d on a G i l f o r d Model 2 4 0 0 r e c o r d i n g s p e c t o p h o t o m e t e r f i t t e d w i t h a r e c i r c u l a t i n g constant temperature water bath. The a s s a y c o n d i t i o n s were t h e same as t h o s e p r e v i o u s l y d e s c r i b e d ( 1 0 ) . T o t a l p l a s m a p r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y t h e b u i r e t method ( 1 6 ) a f t e r c o r r e c t i o n o f p l a s m a h e m o g l o b i n w h i c h was d e t e r m i n e d b y t h e c y a n m e t h e m o g l o b i n method ( 1 5 ) . Plasma o s m o l a l i t y was measured b y a Wescor model 5 1 0 0 v a p o r p r e s s u r e osmometer. S t a t i s t i c a l Methods. Means o f t r e a t m e n t groups f o r p l a s m a r e t e n t i o n o f BSP, p l a s m a o s m o l a l i t y , t o t a l p l a s m a p r o t e i n c o n c e n t r a t i o n and u r i n e f l o w r a t e s were compared b y s t u d e n t s t t e s t f o r i n d e p e n d e n t sample means ( 1 7 ) . P l a s m a enzyme a c t i v i t y d a t a were c o n v e r t e d t o a q u a n t a l f o r m and a n a l y z e d by the F i s c h e r Exact P r o b a b i l i t y Test ( 1 8 ) . Values g r e a t e r t h a n 2 s t a n d a r d d e v i a t i o n s (P < 0 . 0 5 ) f r o m t h e c o n t r o l v a l u e were c h o s e n t o i n d i c a t e a p o s i t i v e r e s p o n s e i n t r e a t e d f i s h . Results Acute L e t h a l i t y . The median l e t h a l dose ( L D Q ) o f MCB t o r a i n b o w t r o u t was e s t i m a t e d t o b e 1 . 8 m l / k g a t 2 4 h . No m o r t a l i t i e s were o b s e r v e d i n a n y o f t h e t r e a t e d a n i m a l s a f t e r t h i s t i m e . The 2 4 h LD50 v a l u e f o r CCI4 was e s t i m a t e d t o be 4 . 7 5 m l / k g b u t , u n l i k e f i s h t r e a t e d w i t h MCB, f i s h i n t o x i c a t e d w i t h CCI4 c o n t i n u e d t o d i e t h r o u g h o u t t h e e n t i r e 9 6 h observation period. 5

E f f e c t o f I n t o x i c a t i o n b y MCB o r CCI4 on P l a s m a C l e a r ance o f BSP and P l a s m a A c t i v i t y o f GPT. S u b l e t h a l doses o f MCB a n d CCI4 were chosen as s i m p l e m u l t i p l e s o f t h o s e u s e d i n mammalian s t u d i e s . F o r MCB t h e doses 0 . 5 m l / k g and 1 . 0 ml/kg r e p r e s e n t e d 0 . 2 8 and 0 . 5 6 r e s p e c t i v e l y o f t h e e s t i m a t e d LD50 v a l u e f o r t h e t r o u t . The d o s e s 1 . 0 m l / k g and 2 . 0 m l / k g u s e d f o r CCI4 r e p r e s e n t . 2 1 and . 4 2 o f i t s e s t i m a t e d LD Q value. F i s h t r e a t e d w i t h MCB t h u s t o l e r a t e d a r e l a t i v e l y g r e a t e r s u b l e t h a l dose o f t o x i c a n t t h a n d i d t h o s e f i s h t r e a t e d w i t h CCI4. Our p r e v i o u s e x p e r i e n c e has i n d i c a t e d t h a t t h i s volume r a n g e i s s u i t a b l e f o r t h e s e t y p e s o f s t u d i e s w i t h t r o u t . A h i g h e r c o n c e n t r a t i o n o f BSP (P < 0 . 0 5 ) was e v i d e n t i n 5


PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS

404

t h e p l a s m a o f f i s h t r e a t e d w i t h 1.0 m l / k g o f MCB o n l y a t 24 h (Fig. 1). No s i g n i f i c a n t e l e v a t i o n o f BSP was f o u n d i n f i s h t r e a t e d w i t h 0.5 m l / k g MCB. E l e v a t e d p l a s m a BSP c o n c e n t r a t i o n s (P < 0.05) were e v i d e n t i n f i s h t r e a t e d w i t h 2.0 m l / k g CCI4 a f t e r 24, 48 and 96 h and i n one g r o u p o f f i s h t r e a t e d w i t h 0 . 2 m l / k g C C 1 a f t e r 24 h . P l a s m a GPT a c t i v i t y v a r i e d g r e a t l y i n a n i m a l s r e c e i v i n g MCB a t 1 m l / k g and was s i g n i f i c a n t l y i n c r e a s e d i n t e s t a n i m a l s o n l y 72 h p o s t - t r e a t m e n t ( F i g . 2 ) . Conversely, p l a s m a GPT a c t i v i t y was e l e v a t e d (P < 0 . 0 1 ) i n g r o u p s o f f i s h r e c e i v i n g e i t h e r 1.0 m l / k g o r 2 . 0 m l / k g o f CCI4 a t b o t h 24 and 48 h . Gross p a t h o l o g i c a l responses o f t h e t r o u t t o t h e t o x i c a n t were c o m p a r a b l e i n f i s h t r e a t e d w i t h e i t h e r MCB o r CCI4. S l i g h t t o moderate h e m o r r h a g i n g , w h i c h p e r s i s t e d t h r o u g h o u t t h e s t u d y , was n o t e d a t t h e b a s e o f b o t h s i n g l e and p a i r e d f i n s a f t e r 12 h i n f i s h t r e a t e d w i t h e i t h e r dose o f MCB o r C C I 4 , however h e m o r r h a g i c a r e a s were o b s e r v e d more c o n s i s t e n t l y i n f i s h t r e a t e d w i t h CCI4. Inspection o f the p e r i t o n e a l c a v i t i e s o f animals t r e a t e d with both chemicals r e v e a l e d areas o f m i l d t o moderate i n f l a m m a t i o n i n s e c t i o n s o f b o t h t h e p e r i t o n e a l l i n i n g s and t h e s m a l l and l a r g e i n t e s tines. The s u r f a c e o f b o t h t h e l i v e r and s p l e e n were m o t t l e d with blanched areas. The l i v e r t o body w e i g h t r a t i o was n e v e r s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s i n any t r e a t m e n t g r o u p . The s p l e e n s o f a n i m a l s t r e a t e d w i t h b o t h d o s e s o f CCI4 and w i t h t h e h i g h dose o f MCB were e n l a r g e d i n t h e m a j o r i t y o f f i s h e x a m i n e d , however wet w e i g h t s o f t h i s o r g a n were n e v e r t a k e n . Unlike c o n t r o l f i s h and t h o s e t r e a t e d w i t h C C I 4 , t h e s u r f a c e o f t h e s p l e e n s f r o m f i s h t r e a t e d w i t h MCB h a d a bumpy, c o a r s e t e x t u r e . H e m o g l o b i n e m i a was p r o n o u n c e d i n f i s h t r e a t e d w i t h b o t h d o s e s o f C C 1 and w i t h t h e h i g h e s t dose o f MCB. A f t e r 24 h t h e mean p l a s m a h e m o g l o b i n c o n c e n t r a t i o n i n a n i m a l s t r e a t e d w i t h 2.0 ml/kg C C 1 g t h a n 200 mg/100 ml w h i l e i n f i s h r e c e i v i n g MCB ( 1 . 0 ml/kg) t h e p l a s m a h e m o g l o b i n c o n c e n t r a t i o n was n o t more t h a n 150 mg/100 m l . H e m o g l o b i n u r i a was e v i d e n t i n f i s h t r e a t e d w i t h CCI4 b u t was n e v e r a p p a r e n t i n MCB t r e a t e d a n i m a l s .


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E f f e c t o f CCI4 I n t o x i c a t i o n on W a t e r B a l a n c e i n Rainbow Trout. B e c a u s e o u r e a r l i e r work h a d i n d i c a t e d t h a t CCI4 i n t o x i c a t i o n r e s u l t e d i n s i g n i f i c a n t weight gain i n s p i n a l t r a n s e c t e d r a i n b o w t r o u t (12) w e i g h t change was d e t e r m i n e d i n i n t a c t f i s h t r e a t e d w i t h MCB o r C C I 4 . The w h o l e body wet w e i g h t o f t r o u t t r e a t e d w i t h MCB d i d n o t change a p p r e c i a b l y f r o m c o n t r o l a n i m a l s a t any t i m e d u r i n g t h e c o u r s e of the i n t o x i c a t i o n (Fig. 3 ) . S i g n i f i c a n t differences (P < .05) were o b s e r v e d i n w h o l e b o d y wet w e i g h t change i n f i s h t r e a t e d w i t h C C 1 a t b o t h 1.0 m l / k g and 2 . 0 m l / k g . F i s h t r e a t e d w i t h CCI4 e i t h e r l o s t l e s s w e i g h t t h a n c o n t r o l 4



24.

WEBER ET AL.

Carbon

Tetrachloride

and

24 48 72

Monochlorobenzene

HOURS:

Pooled 24 4 8 72

Pooled 24

24 48 96

TREATMENT:

Control

MCB

MCB

Control

CCI

DOSE (ml/kg)'

-

0.5

1.0

-

0.2

4

2.0

Figure 1. Phsma BSP concentrations in rainbow trout following single ip injections of either CClj, (0.2 mL/kg or 2.0 mL/kg) or MCB (0.5 mL/kg or 1.0 mL/kg). Plasma dye concentrations were determined 45 min after a single dose of BSP (5.0 mg/kg) was given. Values represent the mean ± SE of the number of animals indicated in parentheses; (*), values which are significantly different from controls.


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PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS


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HOURS:

Pooled

24 48 72

Pooled

TREATMENT:

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MCB

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CCI

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1.0

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DOSE (ml/kg):

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cci

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Figure 2. Phsma GPT activity in rainbow trout following treatment with either CCl (1.0 mL/kg or 2.0 mL/kg) or MCB (0.5 mL/kg or 1.0 mL/kg). Values rep resent the mean ± SE of the number of animals indicated in parentheses; (*), values that are significantly different from controls (*, Ρ < 0.05; **, F < 0.01). h



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Carbon

WEBER ET AL.

Tetrachloride

and

Monochlorobenzene

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4.0 h

Alterations in Rainbow Trout Liver Function and Body Fluids Following

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