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Biotransformation of Selected Chemicals by Fish JOHN L. ALLEN and VERDEL K. DAWSON U.S. Fish and Wildlife Service, National Fishery Research Laboratory, P.O. Box 818, La Crosse, WI 54601 JOSEPH B. HUNN U.S. Fish and Wildlife Service, Hammond Bay Biological Station, Millersburg, MI 49759 F i s h have been used as experimental animals f o r almost 200 years. However, t h e i r status in t h i s r o l e has not always been held in high regard. Huxley (I), speaking through Dr. Obispo, c h a r a c t e r i z e d f i s h as f o l l o w s : "The worst experimental animals in the w o r l d , he s a i d . . . Nobody has a r i g h t to t a l k about t e c h n i c a l d i f f i c u l t i e s who hadn't t r i e d to work with f i s h . Take the simplest o p e r a t i o n ; i t was a nightmare. Had you ever t r i e d to keep i t s g i l l s properly wet while i t was anesthetized on the operating table? Or, a l t e r n a t e l y , to do your surgery under water? Had you ever set out to determine a f i s h ' s basal metabolism, or take an e l e c t r o - c a r d i o g r a m of i t s heart a c t i o n , or measure i t s blood pressure? Had you ever wanted to analyze i t s excreta? And, i f s o , did you know how hard i t was even to c o l l e c t them? Had you ever attempted to study the chemistry of a f i s h ' s d i g e s t i o n and a s s i m i l a t i o n ? To measure i t s speed of i t s nervous reactions? "No, you had not . . . And u n t i l you had, you have no r i g h t to complain about a n y t h i n g . " Despite a l l the problems attendant on studies of a q u a t i c a n i mals, however, great s t r i d e s have been made in the past 10 years in d e f i n i n g biochemical pathways used by f i s h e s to biotransform and e l i m i n a t e x e n o b i o t i c s (2, 3, 4, 5). Many of the e a r l i e r s t u d i e s , e s p e c i a l l y the extensive work of DeWaide (6), defined various biochemical transformations which x e n o b i o t i c s may undergo in v i t r o . Only in the past 10 years have in v i v o studies been undertaken to define the routes and rates of e l i m i n a t i o n of xenob i o t i c s by f i s h e s {7, 8, 9, 10, 11). The studies on which we report here were conducted as part of an ongoing program to evaluate the safety of various chemicals ( a n e s t h e t i c s , c o l l e c t i n g a i d s , s e l e c t i v e t o x i c a n t s , and a h e r b i cide) that are used on f i s h , or that are used in the aquatic environment, or are p o s s i b l e contaminants of that environment. The various biotransformation reactions c h a r a c t e r i z e d here represent only a small f r a c t i o n of the biotransformations that may occur in f i s h . This chapter not subject to U.S. copyright. Published 1979 American Chemical Society Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS
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Glucuronide
Conjugation
The s e l e c t i v e l a m p r i c i d e , 3 ~ t r î f 1 u o r o m e t h y 1 - 4 - n i t r o p h e n o l (TFM), is used to control the sea lamprey (Petromyzon marinus) in the Great Lakes (12, 13). Recent studies have shown that in rats TFM is p r i m a r i l y biotransformed to reduced TFM (14). The major metabolite (Figure 1) found in rainbow trout (Salmo gairdneri), however, was the glucuronide conjugate of TFM (15, 16). The dynamics of TFM and i t s g l u c u r o n i c a c i d conjugate in rainbow trout were reported by Hunn and A l l e n (17). The major increase in the accumulation of TFM conjugate in the b i l e occurred at the same time (between 0.75 and 1.0 h of exposure) that the concentration of conjugate in the plasma dropped. Hunn and A l l e n (18) found that e l i m i n a t i o n of f r e e and c o n j u gated TFM occurred by way of the kidney in coho salmon (Oncorhynchus kisutch) and that conjugated TFM made up the bulk of that excreted. The lampricide r a p i d l y cleared from blood (18, 19) and muscle (20) during recovery in f r e s h water, but the TFM conjugate accumulated in g a l l b l a d d e r b i l e (17, 19). Biotransformation of xenobiotics by f i s h to w a t e r - s o l u b l e conjugates of g l u c u r o n i c a c i d f a c i l i t a t e s b i l i a r y and urinary exc r e t i o n and probably decreases t o x i c i t y (16). Lech and Statham (21) reported that sea lampreys demonstrated a lower rate of glucuronide formation than did rainbow t r o u t , on the basis of in v i t r o glucuronyl t r a n s f e r a s e assays. They a l s o showed that in v i v o sea lampreys had higher c i r c u l a t i n g l e v e l s of free than of conjugated TFM. Pretreatment of sea lampreys and rainbow trout with s a l i c y l a m i d e , an i n h i b i t o r of glucuronyl t r a n s f e r a s e , s h i f t e d the L C f o r trout from 9 · 7 mg/1 to 3.6 mg/1, but did not a l t e r the LC Q f ° lampreys. This s h i f t suggests that glucuronide formation may be the mechanism that provides TFM's s e l e c t i v e toxici ty. Another l a m p r i c i d e , 2 ' , 5 ~ d î c h l o r o - V - n i t r o s a l i c y l a n i 1 i d e (Bayer 73), appears to be eliminated by rainbow trout in the same manner as TFM (22). Statham and Lech (22) observed the presence of a polar metabolite in b i l e ; a n a l y s i s of the metabolite by thin layer chromatography, β - g l u c u r o n i d a s e h y d r o l y s i s , a c i d h y d r o l y s i s , i n f r a r e d spectroscopy, and mass spectrometry indicated that the material was the glucuronide conjugate of Bayer 73 (Figure 1). Schultz and Harman (23) noted that b i l e of largemouth bass (Micropterus sdlmoides) exposed to the lampricide contained the glucuronide conjugate of Bayer 73. A l l e n et a l . (Il) found that Bayer 73 was excreted r e n a l l y by rainbow t r o u t . They recovered 51 yg of the lampricide in the urine a f t e r an i n t r a p e r i t o n e a l i n j e c t i o n of 200 yg of Bayer 73· The a n a l y s i s of urine before and a f t e r β - g l u c u r o n î d a s e incubation and t h i n layer chromatography indicated that most of the material excreted r e n a l l y was the glucuronide conjugate. 5 0
r
s
e
a
5
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
7.
ALLEN ET AL.
Biotransformation
Name
Structure
of
123
Chemicals
Biochemical Pathway and Metabolites Nitro Reduction
Glucuronide Conjugation Q-Glucuronic acid
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TFM 3-trifluoromethyl-4-nitrophenol
Glucuronide Conjugation
Bayer 73 aminoethanol salt of 2,6-dichloro-4-nitroealicylanilide ,
,
O-Glucuronic acid H N-CH -CH OH 2
2
2
Glucuronide Conjugation
Piscaine 2-amino-4-phenyl thiazole
9
C-Ç-H
ρ
C N- C S- H
L. .
N-Acetylation
C-C-H I I N S
C-Ç-H N S
Vo
Y
H-A-C-CH,
H-N-Glucuronic acid
MS-222 methane sulfonate of m-aminobenzoic acid ethyl ester
H N : HSO3CH3 2
N-Acetylatio
Hydrolysis'*' N-Acetylation
f
HN-C-CH,
Ο
H-N-C-
C-OCHc
C-0-C H
2
2
5
N-dealkylation Metabolite HI
Metabolite TL
Dinitramine NJi-diethyl-2,4-dinitro-6trifluoromethyl-m- phenylenediamine 3
3
S-methylation
Thanite isobornyl thiocyanoacetate
Figure 1.
Common name, chemical name, structure, biochemical pathway, and structure of metabolites
Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC ORGANISMS
In a s e r i e s of studies on 2-amino-4-pheny1thiazole (Piscaine), a f i s h a n e s t h e t i c , Suzuki and co-workers (24) i s o l a t e d and i d e n t i f i e d an N-glucuronide metabolite (2-amino-4-pheny1thiazole-2-N-$mono-D-glucopyranosiduronic acid) from medaka (Oryzias latipes), rainbow t r o u t , and carp (Cyprinus earpio) (Figure 1).
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Hydrolysis On the basis of studies on the metabolism of the f i s h anest h e t i c , methane s u l f o n a t e of m-aminobenzoic a c i d ethyl e s t e r (MS-222), by a shark (the spiny d o g f i s h , Squalus acanthias), Maren et a l . (26) reported the formation of a metabolite, m-aminobenzoic a c i d , by cleavage of the e s t e r bond (Figure 1). This e s t e r - h y d r o lyzed product was not p a r t i t i o n e d across the g i l l , but was slowly excreted by the kidney. Luhning (26) measured residues of MS-222 and i t s metabolites in the muscle t i s s u e of s t r i p e d bass (Morone s a x a t i l i s ) , b l u e g i l l s (Lepomis macroohirus), and largemouth bass anesthetized with a 100-mg/l s o l u t i o n of the drug. S t r i p e d bass r a p i d l y hydrolyzed MS-222 to m-aminobenzoic a c i d , but b l u e g i l l s and largemouth bass contained only a small amount of the a c i d r e s i d u e . The concentrat i o n of m-aminobenzoic a c i d residues in s t r i p e d bass muscle t i s s u e continued to increase during a 50-min exposure to MS-222, whereas residues of f r e e MS-222 peaked and d e c l i n e d a f t e r 30 min of exposure. H y d r o l y s i s of MS-222 a l s o occurred during s t o r a g e o f s t r i p e d bass muscle samples, but almost none occurred during storage of b l u e g i l l or largemouth bass samples. S t r i p e d bass apparently possess an esterase not prevalent in b l u e g i l l s or largemouth bass that can hydrolyze the e s t e r linkage of MS-222. Hydrolysis of Bayer 73 was a l s o observed in c a r p , (D. P. Schultz personal communication). He i d e n t i f i e d the h y d r o l y s i s product, 2 - c h l o r o - 4 - n i t r o a n i 1 i n e , in the b i l e of carp exposed to 0.05 mg/1 of Bayer 73 (Figure 1).
Aoetylation F i s h a l s o metabolize x e n o b i o t i c s by N - a c e t y l a t i o n . This process is well documented f o r the f i s h a n e s t h e t i c MS-222 (25-30). Hunn (28) showed that rainbow t r o u t which were anesthetized with MS-222 excreted f r e e and a c e t y l a t e d forms of the drug r e n a l l y (Figure 1). MS-222 injected i n t r a p e r i t o n e a l l y was a l s o excreted in both forms. In both experiments, 77 to 3k% of the MS-222 excreted r e n a l l y was a c e t y l a t e d . In the b l o o d , the major form p r e s ent was f r e e MS-222. Although MS-222 was excreted r e n a l l y , 79 to 85% of the injected dose was excreted extrarena11 y , presumably across the g i l l s . In a s i m i l a r study with the spiny d o g f i s h shark, Maren et a l . (25) reported that 95% of MS-222 was eliminated e s s e n t i a l l y i n t a c t across the g i l l s w i t h i n 2 h a f t e r i n j e c t i o n , but a small percentage (
